Extensive Plasmid Library to Prepare Tau Protein Variants and Study Their Functional Biochemistry

Tau neurofibrillary tangles are key pathological features of Alzheimer’s disease and other tauopathies. Recombinant protein technology is vital for studying the structure and function of tau in physiology and aggregation in pathophysiology. However, open-source and well-characterized plasmids for efficiently expressing and purifying different tau variants are lacking. We generated 44 sequence-verified plasmids including those encoding full length (FL) tau-441, its four-repeat microtubule-binding (K18) fragment, and their respective selected familial pathological variants (N279K, V337M, P301L, C291R, and S356T). Moreover, plasmids for expressing single (C291A), double (C291A/C322A), and triple (C291A/C322A/I260C) cysteine-modified variants were generated to study alterations in cysteine content and locations. Furthermore, protocols for producing representative tau forms were developed. We produced and characterized the aggregation behavior of the triple cysteine-modified tau-K18, often used in real-time cell internalization and aggregation studies because it can be fluorescently labeled on a cysteine outside the microtubule-binding core. Similar to the wild type (WT), triple cysteine-modified tau-K18 aggregated by progressive β-sheet enrichment, albeit at a slower rate. On prolonged incubation, cysteine-modified K18 formed paired helical filaments similar to those in Alzheimer’s disease, sharing morphological phenotypes with WT tau-K18 filaments. Nonetheless, cysteine-modified tau-K18 filaments were significantly shorter (p = 0.002) and mostly wider than WT filaments, explainable by their different principal filament elongation pathways: vertical (end-to-end) and lateral growth for WT and cysteine-modified, respectively. Cysteine rearrangement may therefore induce filament polymorphism. Together, the plasmid library, the protein production methods, and the new insights into cysteine-dependent aggregation should facilitate further studies and the design of antiaggregation agents

Increased plasma neurofilament light chain concentration correlates with severity of post-mortem neurofibrillary tangle pathology and neurodegeneration

Alzheimer's disease (AD) is pathologically characterized by the accumulation of amyloid-β (Aβ) plaques, neurofibrillary tangles and widespread neuronal loss in the brain. In recent years, blood biomarkers have emerged as a realistic prospect to highlight accumulating pathology for secondary prevention trials. Neurofilament light chain (NfL), a marker of axonal degeneration, is robustly elevated in the blood of many neurological and neurodegenerative conditions, including AD. A strong relationship with cerebrospinal fluid (CSF) NfL suggests that these biomarker modalities reflect the same pathological process. Yet, the connection between blood NfL and brain tissue pathology has not been directly compared. In this study, longitudinal plasma NfL from cognitively healthy controls (n = 12) and AD participants (n = 57) were quantified by the Simoa platform. On reaching post-mortem, neuropathological assessment was performed on all participants, with additional frozen and paraffin-embedded tissue acquired from 26 participants for further biochemical (Aβ1-42, Aβ1-40, tau) and histological (NfL) evaluation. Plasma NfL concentrations were significantly increased in AD and correlated with cognitive decline, independent of age. Retrospective stratification based on Braak staging revealed that baseline plasma NfL concentrations were associated with higher neurofibrillary tangle pathology at post-mortem. Longitudinal increases in plasma NfL were observed in all Braak groupings; a significant negative association, however, was found between plasma NfL at time point 1 and both its rate of change and annual percentage increase. Immunohistochemical evaluation of NfL in the medial temporal gyrus (MTG) demonstrated an inverse relationship between Braak stages and NfL staining. Importantly, a significant negative correlation was found between the plasma NfL measurement closest to death and the level of NfL staining in the MTG at post-mortem. For the first time, we demonstrate that plasma NfL associates with the severity of neurofibrillary tangle pathology and neurodegeneration in the post-mortem brain

Statin treatment and beta-Amyloid Production in Patients with Alzheimer s Disease

Alzheimer s disease (AD) is a progressive neurodegenerative disorder pathologically characterized by neurofibrillary tangles and amyloid plaques. According to the amyloid cascade hypothesis, the increased production of beta-amyloid (Abeta), derived form the amyloid precursor protein (APP), and its aggregation into plaques are key pathological events, driving neurodegeneration in AD. This hypothesis is primarily based on disease causing mutations found in familial forms of AD. The sporadic form of AD represents the vast majority of all AD cases. The cause of overproduction of Abeta in the sporadic form of AD, whether there is any, is not known. Epidemiological, clinical and experimental evidence suggests a link between cholesterol and sporadic AD. The hypothesis for the mechanistic link suggests that cholesterol-lowering treatment using statins reduces cholesterol content of cellular membranes. Thereby the non-amyloidogenic pathway, i.e. decreased Abeta production, is supported. The overall aim of this thesis was to evaluate whether cholesterol lowering treatment using statins has any effect on Abeta production in human beings. Efficacy parameters analyzed were markers for cholesterol homeostasis, including cholesterol, lathosterol, 24S-hydroxycholesterol and 27-hydroxycholesterol, and APP metabolism, including alpha-cleaved soluble APP (alphasAPP), betasAPP and total APP. The thesis includes four open clinical studies where we hypothesize that statin treatment will affect cerebrospinal fluid (CSF) and plasma levels of Abeta, and also a fifth exploratory study trying to advance the cholesterol hypothesis by elucidating whether there are alternative mechanisms underlying the link between statins and reduced prevalence of AD. Despite reduced levels of total cholesterol and LDL cholesterol after 12-36 weeks of statin treatment, no effect on Abeta levels were found. After a prolonged treatment period to 12 months, brain cholesterol synthesis was demonstrated to be reduced, but no effect was seen on levels of Abeta1-40/42 in CSF or plasma. This absence of effect on Abeta was further demonstrated when additional Abeta peptides were analyzed, truncated at the C-terminal Abeta1-37/38 and 39. These findings suggest that the cholesterol dependent mechanism for statins in risk-reduction of AD does not seem to affect Abeta production. However, statins have been reported to have other, pleiotropic, effects which are neuroprotective, in addition to inhibiting cholesterol synthesis. In paper V statin treatment was found to produce a significant change in the CSF levels of 44 proteins. Multivariate analyses revealed eight protein changes which, together, provided the best separation of samples taken at baseline and endpoint. One of these proteins, found to be increased after statin treatment, was identified as C3 anaphylatoxin (C3a), a component of the complement system. In conclusion, this thesis does not support the cholesterol hypothesis demonstrated in experimental studies, as valid in patients. We suggest that the anti-inflammatory effect of statin treatment may be part of the underlying link between statin treatment and reduced prevalence of AD

Increase in β-Amyloid Levels in Cerebrospinal Fluid of Children with Down Syndrome

Background: Individuals with Down syndrome (DS) invariably develop Alzheimer’s disease (AD) during their life span. It is therefore of importance to study young DS patients when trying to elucidate early events in AD pathogenesis. Aim: To investigate how levels of different amyloid- _ (A _ ) peptides, as well as tau and phosphorylated tau, in cerebrospinal fluid (CSF) from children with DS change over time. The first CSF sample was taken at 8 months and the following two samples at 20–40 and 54 months of age. Results: Individual levels of the A _ peptides, as well as total A _ levels in CSF increased over time when measured with Western blot. Tau in CSF decreased whereas there was no change in levels of phosphorylated tau over time. Conclusion: The increasing levels of A _ in CSF during early childhood of DS patients observed in this study are probably due to the trisomy of the A _ precursor APP, which leads to an overproduction of A _ . Despite the increased CSF concentrations of A _ , there were no signs of an AD-indicating tau pattern in CSF, since the levels of total tau decreased and phosphorylated tau remained unchanged. This observation further strengthens the theory of A _ pathology preceding tau pathology in AD

Synaptic Molecular and Neurophysiological Markers Are Independent Predictors of Progression in Alzheimer's Disease.

BACKGROUND Cerebrospinal fluid (CSF) neurogranin and quantitative electroencephalography (qEEG) are potential molecular and functional markers of synaptic pathology in Alzheimer's disease (AD). Synaptic markers have emerged as candidate prognostic indicators of AD since synaptic degeneration was shown to be an early event and the best correlate of cognitive deficits in patients along the disease continuum. OBJECTIVE The present study investigated the association between CSF neurogranin and qEEG measures as well as their potential to predict clinical deterioration in mild cognitive impairment (MCI) patients. METHODS Patients diagnosed with MCI (n = 99) underwent CSF conventional AD biomarkers and neurogranin analysis and resting-state EEG recordings. The study population was further stratified into stable (n = 41) and progressive MCI (n = 31), based on the progression to AD dementia during two years follow-up. qEEG analysis included computation of global field power and global field synchronization in four conventional frequency bands. RESULTS CSF neurogranin levels were associated with theta power and synchronization in the progressive MCI group. CSF neurogranin and qEEG measures were significant predictors of progression to AD dementia, independent of baseline amyloid status in MCI patients. A combination of CSF neurogranin with global EEG power in theta and global EEG synchronization in beta band exhibited the highest classification accuracy as compared to either of these markers alone. CONCLUSION qEEG and CSF neurogranin are independent predictors of progression to AD dementia in MCI patients. Molecular and neurophysiological synaptic markers may have additive value in a multimodal diagnostic and prognostic approach to dementia

Update on biomarkers for amyloid pathology in Alzheimer's disease

At the center of Alzheimer's disease pathogenesis is the aberrant aggregation of amyloid-β (Aβ) into oligomers, fibrils and plaques. Effective monitoring of Aβ deposition directly in patients is essential to assist anti-Aβ therapeutics in target engagement and participant selection. In the advent of approved anti-Aβ therapeutics, biomarkers will become of fundamental importance in initiating treatments having disease modifying effects at the earliest stage. Two well-established Aβ biomarkers are widely utilized: Aβ-binding ligands for positron emission tomography and immunoassays to measure Aβ42 in cerebrospinal fluid. In this review, we will discuss the current clinical, diagnostic and research state of biomarkers for Aβ pathology. Furthermore, we will explore the current application of blood-based markers to assess Aβ pathology

Neurofilaments can differentiate ALS subgroups and ALS from common diagnostic mimics

Delayed diagnosis and misdiagnosis are frequent in people with amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease (MND). Neurofilament light chain (NFL) and phosphorylated neurofilament heavy chain (pNFH) are elevated in ALS patients. We retrospectively quantified cerebrospinal fluid (CSF) NFL, CSF pNFH and plasma NFL in stored samples that were collected at the diagnostic work-up of ALS patients (n = 234), ALS mimics (n = 44) and controls (n = 9). We assessed the diagnostic performance, prognostication value and relationship to the site of onset and genotype. CSF NFL, CSF pNFH and plasma NFL levels were significantly increased in ALS patients compared to patients with neuropathies & myelopathies, patients with myopathies and controls. Furthermore, CSF pNFH and plasma NFL levels were significantly higher in ALS patients than in patients with other MNDs. Bulbar onset ALS patients had significantly higher plasma NFL levels than spinal onset ALS patients. ALS patients with C9orf72HRE mutations had significantly higher plasma NFL levels than patients with SOD1 mutations. Survival was negatively correlated with all three biomarkers. Receiver operating characteristics showed the highest area under the curve for CSF pNFH for differentiating ALS from ALS mimics and for plasma NFL for estimating ALS short and long survival. All three biomarkers have diagnostic value in differentiating ALS from clinically relevant ALS mimics. Plasma NFL levels can be used to differentiate between clinical and genetic ALS subgroups