University College London/University of Gothenburg PhD course "Biomarkers in neurodegenerative diseases" 2019—course organisation

Biomarkers are increasingly employed for effective research into neurodegenerative diseases. They have become essential for reaching an accurate clinical diagnosis, monitoring disease, and refining entry criteria for participation in clinical treatment trials, and will be key in measuring target engagement and treatment outcome in disease-modifying therapies. Emerging techniques and research combining different biomarker modalities continue to strengthen our understanding of the underlying pathology and the sequence of pathogenic events. Given recent advances, we are now at a pivotal stage in biomarker research. PhD students working in the field of neurodegenerative disease require a working knowledge of a range of biomarkers available and their limitations, to correctly interpret scientific literature and to design and conduct successful research studies themselves. Here, we outline the University College London/University of Gothenburg “Biomarkers in neurodegenerative diseases course”, the first initiative of its kind aimed to bring together both experts and PhD students from all areas within the field of neurodegeneration, to provide comprehensive knowledge of biomarker research for the next generation of scientists

Cerebrospinal fluid biomarkers in Alzheimer’s Disease: from bedside to bench and back

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that results in cognitive impairment and death. The pathological hallmarks are extracellular cortical amyloid plaques and intraneuronal tangles composed of hyperphosphorylated tau. Although environmental and genetic factors contribute to the development of AD, the sequence of pathophysiological events that lead to Alzheimer’s dementia is not yet completely clear. The clinical diagnosis of AD during life can be challenging and factors that explain clinical phenotypic heterogeneity and variability in rates of disease progression are not well understood. Biomarkers, objective measures of biological function, can be employed to support a clinical diagnosis of AD and may be abnormal before the onset of clinical symptoms. Imaging and cerebrospinal fluid biomarkers (CSF) are now incorporated into clinical and research diagnostic criteria. CSF, which is in direct contact with the brain, is a promising source of biomarkers and has the potential to differentiate AD from other neurodegenerative dementias, explain clinical heterogeneity within AD and elucidate the role of other pathobiological pathways. Ultimately CSF biomarkers might facilitate diagnosis of AD in its pre-clinical phase and allow for treatment responses to be measured. In this thesis CSF samples from clinical cohorts of individuals with AD, other neurodegenerative diseases and healthy controls are analysed using an extended panel of enzyme-linked immunosorbent assays (ELISA) and a novel mass spectrometry based assay. For the established CSF biomarkers, the practical issues related to collection, transportation and storage of CSF are investigated. Amyloid positron emission tomography (PET) imaging is investigated as a means of validating clinical cutpoints. An extended panel of established and emerging ELISAs is used to determine the diagnostic utility of biomarkers for differentiating AD from other neurodegenerative dementias and for explaining phenotypic heterogeneity within AD. The role of CSF biomarkers as predictors of disease progression is investigated employing robust measures of brain atrophy as surrogate measures of rates of neurodegeneration. Finally CSF samples are probed for new AD biomarkers using a novel mass spectrometry based assay. A number of practical conclusions are drawn from this work: aliquot storage volume is identified as an important confounder in measured CSF b-Amyloid concentration. CSF laboratory transportation methods are shown not to have a significant impact on measured biomarker concentration. Amyloid PET is a valuable means of validating clinical diagnostic cutpoints of core CSF biomarkers. Tau/Ab1-42 ratio, Ab40/42 ratio, P-tau and NFL emerge as having diagnostic utility for differentiating AD from other neurodegenerative diseases, and have high sensitivity and specificity for distinguishing AD from bvFTD, SD and healthy controls. Important differences in T-tau, P-tau and neurofilament light distinguish different AD atypical phenotypes and may help to elucidate underlying biological differences between these syndromes: individuals with the visual variant of AD (posterior cortical atrophy) have the lowest levels of CSF Tau and lowest rates of cognitive decline while the frontal executive cases have highest levels of NFL and highest rates of cognitive decline indicating more rapid neurodegeneration. Several novel biomarkers including trefoil factor 3 and several markers involved in vascular remodeling, amyloid processing and neuroinflammation are identified as predictors of increased atrophy rates in amyloid positive individuals suggesting possible independent mechanisms driving differing rates of neurodegeneration between individuals. Other novel AD biomarkers including malate dehydrogenase are identified as distinguishing AD from controls using a novel mass spectrometry based assay. Moreover, this assay demonstrates how mass spectrometry might be used for biomarker discovery and rapid development of a high throughput multiplexed clinical CSF assay. Taken together these results address some of the unanswered questions about how CSF should be collected, handled and stored to optimize analytical standardization, and how clinical results might be validated using amyloid PET. This work establishes the clinical utility of established biomarkers for differentiating AD from other neurodegenerative diseases and identifies established and novel biomarkers that might explain clinical heterogeneity and rates of progression between individuals. Finally a method for rapidly developing new biomarkers is tested and validated

Mass spectrometry analysis of tau and amyloid-beta in iPSC-derived models of Alzheimer's disease and dementia

Induced pluripotent stem cell (iPSC) technology enables the generation of human neurons in vitro, which contain the precise genome of the cell donor, therefore permitting the generation of disease models from individuals with a disease-associated genotype of interest. This approach has been extensively used to model inherited forms of Alzheimer's disease and frontotemporal dementia. The combination of iPSC-derived neuronal models with targeted mass spectrometry analysis has provided unprecedented insights into the regulation of specific proteins in human neuronal physiology and pathology. For example enabling investigations into tau and APP/Aβ, specifically: protein isoform expression, relative levels of cleavage fragments, aggregated species and functionally critical post-translational modifications. The use of mass spectrometry has enabled a determination of how closely iPSC-derived models recapitulate disease profiles observed in the human brain. This review will highlight the progress to date in studies using iPSCs and mass spectrometry to model Alzheimer's disease and dementia. We go on to convey our optimism, as studies in the near future will make use of this precedent, together with novel techniques such as genome editing and stable isotope labelling, to provide real progress towards an in depth understanding of early neurodegenerative processes and development of novel therapeutic agents

Temporal dynamism of resource capture: a missing factor in ecology?

Temporal dynamism of plant resource capture, and its impacts on plant–plant interactions, can have important regulatory roles in multispecies communities. For example, by modifying resource acquisition timing, plants might reduce competition and promote their coexistence. However, despite the potential wide ecological relevance of this topic, short-term (within growing season) temporal dynamism has been overlooked. This is partially a consequence of historic reliance on measures made at single points in time. We propose that with current technological advances this is a golden opportunity to study within growing season temporal dynamism of resource capture by plants in highly informative ways. We set out here an agenda for future developments in this research field, and explore how new technologies can deliver this agenda

Amyloid β peptides are differentially vulnerable to preanalytical surface exposure, an effect incompletely mitigated by the use of ratios

INTRODUCTION: We tested the hypothesis that the amyloid β (Aβ) peptide ratios are more stable than Aβ42 alone when biofluids are exposed to two preanalytical conditions known to modify measurable Aβ concentration. METHODS: Human cerebrospinal fluid (CSF) and culture media (CM) from human cortical neurons were exposed to a series of volumes and polypropylene surfaces. Aβ42, Aβ40, and Aβ38 peptide concentrations were measured using a multiplexed electrochemiluminescence immunoassay. Data were analyzed using mixed models in R. RESULTS: Decrease of measurable Aβ peptide concentrations was exaggerated in longer peptides, affecting the Aβ42:Aβ40 and Aβ42:Aβ38 ratios. However, the effect size of surface treatment was reduced in Aβ peptide ratios versus Aβ42 alone. For Aβ42:Aβ40, the effect was reduced by approximately 50% (volume) and 75% (transfer) as compared to Aβ42 alone. DISCUSSION: Use of Aβ ratios, in conjunction with concentrations, may mitigate confounding factors and assist the clinical diagnostic process for Alzheimer's disease

Cerebrospinal fluid markers including trefoil factor 3 are associated with neurodegeneration in amyloid-positive individuals.

We aimed to identify cerebrospinal fluid (CSF) biomarkers associated with neurodegeneration in individuals with and without CSF evidence of Alzheimer pathology. We investigated 287 Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects (age=74.9±6.9; 22/48/30% with Alzheimer's disease/mild cognitive impairment/controls) with CSF multiplex analyte data and serial volumetric MRI. We calculated brain and hippocampal atrophy rates, ventricular expansion and Mini Mental State Examination decline. We used false discovery rate corrected regression analyses to assess associations between CSF variables and atrophy rates in individuals with and without amyloid pathology, adjusting in stages for tau, baseline volume, p-tau, age, sex, ApoE4 status and diagnosis. Analytes showing statistically significant independent relationships were entered into reverse stepwise analyses. Adjusting for tau, baseline volume, p-tau, age, sex and ApoE4, 4/83 analytes were significantly independently associated with brain atrophy rate, 1/83 with ventricular expansion and 2/83 with hippocampal atrophy. The strongest CSF predictor for the three atrophy measures was low trefoil factor 3 (TFF3). High cystatin C (CysC) was associated with higher whole brain atrophy and hippocampal atrophy rates. Lower levels of vascular endothelial growth factor and chromogranin A (CrA) were associated with higher whole brain atrophy. In exploratory reverse stepwise analyses, lower TFF3 was associated with higher rates of whole brain, hippocampal atrophy and ventricular expansion. Lower levels of CrA were associated with higher whole brain atrophy rate. The relationship between low TFF3 and increased hippocampal atrophy rate remained after adjustment for diagnosis. We identified a series of CSF markers that are independently associated with rate of neurodegeneration in amyloid-positive individuals. TFF3, a substrate for NOTCH processing may be an important biomarker of neurodegeneration across the Alzheimer spectrum

Music Perception in Dementia.

Despite much recent interest in music and dementia, music perception has not been widely studied across dementia syndromes using an information processing approach. Here we addressed this issue in a cohort of 30 patients representing major dementia syndromes of typical Alzheimer's disease (AD, n = 16), logopenic aphasia (LPA, an Alzheimer variant syndrome; n = 5), and progressive nonfluent aphasia (PNFA; n = 9) in relation to 19 healthy age-matched individuals. We designed a novel neuropsychological battery to assess perception of musical patterns in the dimensions of pitch and temporal information (requiring detection of notes that deviated from the established pattern based on local or global sequence features) and musical scene analysis (requiring detection of a familiar tune within polyphonic harmony). Performance on these tests was referenced to generic auditory (timbral) deviance detection and recognition of familiar tunes and adjusted for general auditory working memory performance. Relative to healthy controls, patients with AD and LPA had group-level deficits of global pitch (melody contour) processing while patients with PNFA as a group had deficits of local (interval) as well as global pitch processing. There was substantial individual variation within syndromic groups. Taking working memory performance into account, no specific deficits of musical temporal processing, timbre processing, musical scene analysis, or tune recognition were identified. The findings suggest that particular aspects of music perception such as pitch pattern analysis may open a window on the processing of information streams in major dementia syndromes. The potential selectivity of musical deficits for particular dementia syndromes and particular dimensions of processing warrants further systematic investigation

Loss and dispersion of superficial white matter in Alzheimer's disease: a diffusion MRI study

Pathological cerebral white matter changes in Alzheimer’s disease have been shown using diffusion tensor imaging. Superficial white matter changes are relatively understudied despite their importance in cortico-cortical connections. Measuring superficial white matter degeneration using diffusion tensor imaging is challenging due to its complex organizational structure and proximity to the cortex. To overcome this, we investigated diffusion MRI changes in young-onset Alzheimer’s disease using standard diffusion tensor imaging and Neurite Orientation Dispersion and Density Imaging to distinguish between disease-related changes that are degenerative (e.g. loss of myelinated fibres) and organizational (e.g. increased fibre dispersion). Twenty-nine young-onset Alzheimer’s disease patients and 22 healthy controls had both single-shell and multi-shell diffusion MRI. We calculated fractional anisotropy, mean diffusivity, neurite density index, orientation dispersion index and tissue fraction (1-free water fraction). Diffusion metrics were sampled in 15 a priori regions of interest at four points along the cortical profile: cortical grey matter, grey/white boundary, superficial white matter (1 mm below grey/white boundary) and superficial/deeper white matter (2 mm below grey/white boundary). To estimate cross-sectional group differences, we used average marginal effects from linear mixed effect models of participants’ diffusion metrics along the cortical profile. The superficial white matter of young-onset Alzheimer’s disease individuals had lower neurite density index compared to controls in five regions (superior and inferior parietal, precuneus, entorhinal and parahippocampus) (all P < 0.05), and higher orientation dispersion index in three regions (fusiform, entorhinal and parahippocampus) (all P < 0.05). Young-onset Alzheimer’s disease individuals had lower fractional anisotropy in the entorhinal and parahippocampus regions (both P < 0.05) and higher fractional anisotropy within the postcentral region (P < 0.05). Mean diffusivity was higher in the young-onset Alzheimer’s disease group in the parahippocampal region (P < 0.05) and lower in the postcentral, precentral and superior temporal regions (all P < 0.05). In the overlying grey matter, disease-related changes were largely consistent with superficial white matter findings when using neurite density index and fractional anisotropy, but appeared at odds with orientation dispersion and mean diffusivity. Tissue fraction was significantly lower across all grey matter regions in young-onset Alzheimer’s disease individuals (all P < 0.001) but group differences reduced in magnitude and coverage when moving towards the superficial white matter. These results show that microstructural changes occur within superficial white matter and along the cortical profile in individuals with young-onset Alzheimer’s disease. Lower neurite density and higher orientation dispersion suggests underlying fibres undergo neurodegeneration and organizational changes, two effects previously indiscernible using standard diffusion tensor metrics in superficial white matter

Diagnosing Dementia in the Clinical Setting: Can Amyloid PET Provide Additional Value Over Cerebrospinal Fluid?

Cerebrospinal fluid (CSF) measures of amyloid and tau are the first-line Alzheimer's disease biomarkers in many clinical centers. We assessed if and when the addition of amyloid PET following CSF measurements provides added diagnostic value. Twenty patients from a cognitive clinic, who had undergone detailed assessment including CSF measures, went on to have amyloid PET. The treating neurologist's working diagnosis, and degree of diagnostic certainty, was assessed both before and after the PET. Amyloid PET changed the diagnosis in 7/20 cases. Amyloid PET can provide added diagnostic value, particularly in young-onset, atypical dementias, where CSF results are borderline and diagnostic uncertainty remains