The phosphorylation cascade hypothesis of Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by amyloid-β (Aβ)-induced phosphorylation of the axon-stabilizing tau protein, which causes neurodegeneration. Here, Morshed et al. show that deregulated phosphorylation in AD also affects other proteins and cell types in the brain, suggesting that the tau-centric view on Aβ toxicity should be revised

Refining the amyloid β peptide and oligomer fingerprint ambiguities in Alzheimer’s disease: Mass spectrometric molecular characterization in brain, cerebrospinal fluid, blood, and plasma

Since its discovery, amyloid-β (Aβ) has been the principal target of investigation of in Alzheimer's disease (AD). Over the years however, no clear correlation was found between the Aβ plaque burden and location, and AD associated neurodegeneration and cognitive decline. Instead, diagnostic potential of specific Aβ peptides and/or their ratio, was established. For instance, a selective reduction of the concentration of the aggregation-prone 42 amino acid-long Aβ peptide (Aβ42) in cerebrospinal fluid (CSF) was put forward as reflective of Aβ peptide aggregation in the brain. With time, Aβ oligomers - the proposed toxic Aβ intermediates - have emerged as potential drivers of synaptic dysfunction and neurodegeneration in the disease process. Oligomers are commonly agreed upon to come in different shapes and sizes, and are very poorly characterized when it comes to their composition and their "toxic" properties. The concept of structural polymorphism - a diversity in conformational organization of amyloid aggregates - that depends on the Aβ peptide backbone, makes characterization of Aβ aggregates and their role in AD progression challenging. In this review, we revisit the history of Aβ discovery and initial characterization and highlight the crucial role mass spectrometry (MS) has played in this process. We critically review the common knowledge gaps in the molecular identity of the Aβ peptide, and how MS is aiding characterization of higher order Aβ assemblies. Finally, we go on to presenting recent advances in MS approaches for characterization of Aβ as single peptides and oligomers, and convey our optimism, as to how MS holds a promise for paving the way for progress towards a more comprehensive understanding of Aβ in AD research

Endo-lysosomal proteins and ubiquitin CSF concentrations in Alzheimer's and Parkinson's disease

BACKGROUND: Increasing evidence implicates dysfunctional proteostasis and the involvement of the autophagic and endo-lysosomal system and the ubiquitin-proteasome system in neurodegenerative diseases. In Alzheimer's disease (AD), there is an accumulation of autophagic vacuoles within the neurons. In Parkinson's disease (PD), susceptibility has been linked to genes encoding proteins involved in autophagy and lysosomal function, as well as mutations causing lysosomal disorders. Furthermore, both diseases are characterized by the accumulation of protein aggregates. METHODS: Proteins associated with endocytosis, lysosomal function, and the ubiquitin-proteasome system were identified in the cerebrospinal fluid (CSF) and targeted by combining solid-phase extraction and parallel reaction monitoring mass spectrometry. In total, 50 peptides from 18 proteins were quantified in three cross-sectional cohorts including AD (N = 61), PD (N = 21), prodromal AD (N = 10), stable mild cognitive impairment (N = 15), and controls (N = 68). RESULTS: A pilot study, including subjects selected based on their AD CSF core biomarker concentrations, showed increased concentrations of several targeted proteins in subjects with core biomarker levels indicating AD pathology compared to controls. Next, in a clinically characterized cohort, lower concentrations in CSF of proteins in PD were found compared to subjects with prodromal AD. Further investigation in an additional clinical study again revealed lower concentrations in CSF of proteins in PD compared to controls and AD. CONCLUSION: In summary, significantly different peptide CSF concentrations were identified from proteins AP2B1, C9, CTSB, CTSF, GM2A, LAMP1, LAMP2, TCN2, and ubiquitin. Proteins found to have altered concentrations in more than one study were AP2B1, CTSB, CTSF, GM2A, LAMP2, and ubiquitin. Interestingly, given the genetic implication of lysosomal function in PD, we did identify the CSF concentrations of CTSB, CTSF, GM2A, and LAMP2 to be altered. However, we also found differences in proteins associated with endocytosis (AP2B1) and the ubiquitin-proteasome system (ubiquitin). No difference in any peptide CSF concentration was found in clinically characterized subjects with AD compared to controls. In conclusion, CSF analyses of subjects with PD suggest a general lysosomal dysfunction, which resonates well with recent genetic findings, while such changes are minor or absent in AD

Amyloid pathology and synaptic loss in pathological aging

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory dysfunction and cognitive decline. Pathological aging (PA) describes patients who are amyloid-positive but cognitively unimpaired at time of death. Both AD and PA contain amyloid plaques dominated by amyloid β (Aβ) peptides. In this study, we investigated and compared synaptic protein levels, amyloid plaque load, and Aβ peptide patterns between AD and PA. Two cohorts of post-mortem brain tissue were investigated. In the first, consisting of controls, PA, AD, and familial AD (FAD) individuals, synaptic proteins extracted with tris(hydroxymethyl)aminomethane-buffered saline (TBS) were analyzed. In the second, consisting of tissue from AD and PA patients from three different regions (occipital lobe, frontal lobe, and cerebellum), a two-step extraction was performed. Five synaptic proteins were extracted using TBS, and from the remaining portion Aβ peptides were extracted using formic acid. Subsequently, immunoprecipitation with several antibodies targeting different proteins/peptides was performed for both fractions, which were subsequently analyzed by mass spectrometry. The levels of synaptic proteins were lower in AD (and FAD) compared with PA (and controls), confirming synaptic loss in AD patients. The amyloid plaque load was increased in AD compared with PA, and the relative amount of Aβ40 was higher in AD while for Aβ42 it was higher in PA. In AD loss of synaptic function was associated with increased plaque load and increased amounts of Aβ40 compared with PA cases, suggesting that synaptic function is preserved in PA cases even in the presence of Aβ

Targeting LAMP2 in human cerebrospinal fluid with a combination of immunopurification and high resolution parallel reaction monitoring mass spectrometry

Background: Alzheimer’s disease is the most common form of dementia. An increasing body of evidence suggests that endo-lysosomal dysfunction is a pathogenic mechanism of Alzheimer’s disease. Thus there is a potential for proteins involved in the normal function of endo-lysosomal vesicles to act as biomarkers of disease. Herein we focused on the lysosomal protein LAMP2 that is involved in chaperone mediated autophagy. / Results: Using a combination of immunoprecipitation, digestion and nano-liquid chromatography tandem mass spectrometry we targeted and identified six tryptic LAMP2 peptides in human cerebrospinal fluid. Employing the identified proteotypic tryptic peptides a hybrid immunoprecipitation high resolution parallel reaction monitoring mass spectrometric method was developed for the relative quantitation of LAMP2. The method was evaluated in a number of experiments which defined the overall methodological as well as the analytical micro-liquid chromatography mass spectrometric intra- and inter-day variability. We identified an overall methodological peptide dependent intra-day variability of 8–16 %. The inter-day experiments showed similar results. The analytical contribution to the variation was minor with a coefficient of variation of 0.5–2.1 %, depending on the peptide. Using the developed method, with defined and limited variability, we report increased cerebrospinal fluid levels of three LAMP2 peptides in Alzheimer’s disease subjects (n = 14), as compared to non-Alzheimer’s disease controls (n = 14). / Conclusion: Altered LAMP2 levels in cerebrospinal fluid may indicate endo-lysosomal dysfunction in Alzheimer’s disease. However, further studies in larger cohorts comprised of well-defined patient materials are required. We here present a tool which can be used for exploring the relevance of the level of LAMP2 as a potential measure of lysosomal dysfunction in Alzheimer’s disease or other neurodegenerative diseases

Quantification of the trans-synaptic partners neurexin-neuroligin in CSF of neurodegenerative diseases by parallel reaction monitoring mass spectrometry

BACKGROUND: Synaptic proteins are increasingly studied as biomarkers for synaptic dysfunction and loss, which are early and central events in Alzheimer's disease (AD) and strongly correlate with the degree of cognitive decline. In this study, we specifically investigated the synaptic binding partners neurexin (NRXN) and neuroligin (Nlgn) proteins, to assess their biomarker's potential. METHODS: we developed a parallel reaction monitoring mass spectrometric method for the simultaneous quantification of NRXNs and Nlgns in cerebrospinal fluid (CSF) of neurodegenerative diseases, focusing on AD. Specifically, NRXN-1α, NRXN-1β, NRXN-2α, NRXN-3α and Nlgn1, Nlgn2, Nlgn3 and Nlgn4 proteins were targeted. FINDINGS: The proteins were investigated in a clinical cohort including CSF from controls (n=22), mild cognitive impairment (MCI) due to AD (n=44), MCI due to other conditions (n=46), AD (n=77) and a group of non-AD dementia (n=28). No difference in levels of NRXNs and Nlgns was found between AD (both at dementia and MCI stages) or controls or the non-AD dementia group for any of the targeted proteins. NRXN and Nlgn proteins correlated strongly with each other, but only a weak correlation with the AD core biomarkers and the synaptic biomarkers neurogranin and growth-associated protein 43, was found, possibly reflecting different pathogenic processing at the synapse. INTERPRETATION: we conclude that NRXN and Nlgn proteins do not represent suitable biomarkers for synaptic pathology in AD. The panel developed here could aid in future investigations of the potential involvement of NRXNs and Nlgns in synaptic dysfunction in other disorders of the central nervous system. FUNDING: a full list of funding can be found under the acknowledgments section

A Parallel Reaction Monitoring Mass Spectrometric Method for Analysis of Potential CSF Biomarkers for Alzheimer's Disease

Scope: The aim of this study was to develop and evaluate a parallel reactionmonitoring mass spectrometry (PRM-MS) assay consisting of a panel ofpotential protein biomarkers in cerebrospinal fluid (CSF).Experimental design: Thirteen proteins were selected based on theirassociation with neurodegenerative diseases and involvement in synapticfunction, secretory vesicle function, or innate immune system. CSF sampleswere digested and two to three peptides per protein were quantified usingstable isotope-labeled peptide standards.Results: Coefficients of variation were generally below 15%. Clinicalevaluation was performed on a cohort of 10 patients with Alzheimer’s disease(AD) and 15 healthy subjects. Investigated proteins of the granin familyexhibited the largest difference between the patient groups. Secretogranin-2(p<0.005) and neurosecretory protein VGF (p<0.001) concentrations werelowered in AD. For chromogranin A, two of three peptides had significantlylowered AD concentrations (p<0.01). The concentrations of the synapticproteins neurexin-1 and neuronal pentraxin-1, as well as neurofascin werealso significantly lowered in AD (p<0.05). The other investigated proteins,β2-microglobulin, cystatin C, amyloid precursor protein, lysozyme C,neurexin-2, neurexin-3, and neurocan core protein, were not significantlyaltered.Conclusion and clinical relevance: PRM-MS of protein panels is a valuabletool to evaluate biomarker candidates for neurodegenerative disorders

Proteomic studies of cerebrospinal fluid biomarkers of Alzheimer's disease: an update

Introduction: Alzheimer’s disease (AD) is a neurodegenerative disease affecting the brain. Today there are three cerebrospinal fluid (CSF) biomarkers, amyloid-β consisting of 42 amino acids (Aβ42), total-tau (t-tau) and phosphorylated-tau (p-tau), which combined have sensitivity and specificity figures around 80%. However, pathological studies have shown that comorbidity is a common feature in AD and that the three currently used CSF biomarkers do not optimally reflect the activity of the disease process. Thus, additional markers are needed. Areas covered: In the present review, we screened PubMed for articles published the last five years (2012–2017) for proteomic studies in CSF with the criteria that AD had to be included as one of the diagnostic groups. Based on inclusion criteria, 28 papers were included reporting in total 224 biomarker-data that were altered in AD compared to control. Both mass spectrometry and multi-panel immunoassays were considered as proteomic studies. Expert commentary: A large number of pilot studies have been reported but so far there is a lack of replicated findings and to date no CSF biomarker discovered in proteomic studies has reached the clinic to aid in the diagnostic work-up of patients with cognitive impairment

Lumbar and ventricular CSF concentrations of extracellular matrix proteins before and after shunt surgery in idiopathic normal pressure hydrocephalus

Background: Idiopathic normal pressure hydrocephalus (iNPH) is a reversible CNS disease characterized by disturbed cerebrospinal fluid (CSF) dynamics. Changes in the extracellular matrix (ECM) composition might be involved in the pathophysiology of iNPH. The aim of this study was to explore possible differences between lumbar and ventricular CSF concentrations of the ECM markers brevican and neurocan, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and their relation to clinical symptoms in iNPH patients before and after shunt surgery. Methods: Paired lumbar and ventricular CSF was collected from 31 iNPH patients, before and four months after shunt surgery. CSF was analysed for concentrations of tryptic peptides originating from brevican and neurocan using a mass spectrometry-based panel, and for MMP-1, -2, -9, -10 and TIMP-1 using fluorescent or electrochemiluminescent immunoassays. Results: Brevican and neurocan peptide levels were not influenced by CSF origin, but MMP-1, -2, -10 and TIMP-1 were increased (p ≤ 0.0005), and MMP-9 decreased (p ≤ 0.0003) in lumbar CSF compared with ventricular CSF. There was a general trend of ECM proteins to increase following shunt surgery. Ventricular TIMP-1 was inversely correlated with overall symptoms (rho = − 0.62, p < 0.0001). CSF concentrations of the majority of brevican and neurocan peptides were increased in iNPH patients with a history of cardiovascular disease (p ≤ 0.001, AUC = 0.84–0.94) compared with those without. Conclusion: Levels of the CNS-specific proteins brevican and neurocan did not differ between the lumbar and ventricular CSF, whereas the increase of several CNS-unspecific MMPs and TIMP-1 in lumbar CSF suggests contribution from peripheral tissues. The increase of ECM proteins in CSF following shunt surgery could indicate disturbed ECM dynamics in iNPH that are restored by restitution of CSF dynamics

Tauopathy-Associated Tau Fragment Ending at Amino Acid 224 Is Generated by Calpain-2 Cleavage

BACKGROUND: Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD. OBJECTIVE: Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224. METHODS: Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y). RESULTS: Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media. CONCLUSIONS: Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged