Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer's disease.

Neurodegenerative diseases are a growing burden, and there is an urgent need for better biomarkers for diagnosis, prognosis, and treatment efficacy. Structural and functional brain alterations are reflected in the protein composition of cerebrospinal fluid (CSF). Alzheimer's disease (AD) patients have higher CSF levels of tau, but we lack knowledge of systems-wide changes of CSF protein levels that accompany AD. Here, we present a highly reproducible mass spectrometry (MS)-based proteomics workflow for the in-depth analysis of CSF from minimal sample amounts. From three independent studies (197 individuals), we characterize differences in proteins by AD status (> 1,000 proteins, CV < 20%). Proteins with previous links to neurodegeneration such as tau, SOD1, and PARK7 differed most strongly by AD status, providing strong positive controls for our approach. CSF proteome changes in Alzheimer's disease prove to be widespread and often correlated with tau concentrations. Our unbiased screen also reveals a consistent glycolytic signature across our cohorts and a recent study. Machine learning suggests clinical utility of this proteomic signature.

Value of a Panel of 6 Serum Biomarkers to Differentiate Between Healthy Controls and Mild Cognitive Impairment Due to Alzheimer Disease.

BACKGROUND: There is considerable evidence suggesting that inflammatory responses may be involved in the neurodegenerative cascade of Alzheimer disease (AD). Blood-based biomarker analysis of inflammatory markers indicative of dementia could serve as a minimally invasive and easy-to-administer diagnostic tool in primary care. MATERIAL AND METHODS: The authors quantified 6 markers (brain-derived neurotrophic factor, insulin-like growth factor 1, vascular endothelial growth factor, transforming growth factor-beta type 1, monocyte chemoattractant protein 1, and interleukin-18) in blood serum of 68 healthy blood donors (controls), 42 patients with AD at the dementia stage, 55 patients with AD at the stage of mild cognitive impairment (MCI-AD), and 25 patients with MCI non-AD. All patients have been fully characterized, including AD biomarker analyses in cerebrospinal fluid. Data were analyzed in an algorithm that was trained, validated, and then used for dichotomous classification of unknown data into data sets suspicious and not suspicious of AD. RESULTS: Using this algorithm, 47 of 55 MCI-AD (85.5%) and 20 of 25 MCI non-AD (80%) cases were classified as suspicious of AD. CONCLUSIONS: This panel of 6 markers in blood serum may indicate underlying neurodegenerative processes in patients with AD at the MCI stage. The authors assume that a deranged equilibrium of neuroprotective and inflammatory processes is an overall major cause for neurodegeneration and cognitive decline.

Apolipoprotein E4 disrupts the neuroprotective action of sortilin in neuronal lipid metabolism and endocannabinoid signaling.

INTRODUCTION: Apolipoprotein E (apoE) is a carrier for brain lipids and the most important genetic risk factor for Alzheimer's disease (AD). ApoE binds the receptor sortilin, which mediates uptake of apoE-bound cargo into neurons. The significance of this uptake route for brain lipid homeostasis and AD risk seen with apoE4, but not apoE3, remains unresolved. METHODS: Combining neurolipidomics in patient specimens with functional studies in mouse models, we interrogated apoE isoform-specific functions for sortilin in brain lipid metabolism and AD. RESULTS: Sortilin directs the uptake and conversion of polyunsaturated fatty acids into endocannabinoids, lipid-based neurotransmitters that act through nuclear receptors to sustain neuroprotective gene expression in the brain. This sortilin function requires apoE3, but is disrupted by binding of apoE4, compromising neuronal endocannabinoid metabolism and action. DISCUSSION: We uncovered the significance of neuronal apoE receptor sortilin in facilitating neuroprotective actions of brain lipids, and its relevance for AD risk seen with apoE4.

The Cerebrospinal Fluid Free-Glycans Hex1 and HexNAc1Hex1Neu5Ac1 as Potential Biomarkers of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting a growing number of elderly people. In order to improve the early and differential diagnosis of AD, better biomarkers are needed. Glycosylation is a protein post-translational modification that is modulated in the course of many diseases, including neurodegeneration. Aiming to improve AD diagnosis and differential diagnosis through glycan analytics methods, we report the glycoprotein glycome of cerebrospinal fluid (CSF) isolated from a total study cohort of 262 subjects. The study cohort consisted of patients with AD, healthy controls and patients suffering from other types of dementia. CSF free-glycans were also isolated and analyzed in this study, and the results reported for the first time the presence of 19 free glycans in this body fluid. The free-glycans consisted of complete or truncated N-/O-glycans as well as free monosaccharides. The free-glycans Hex1 and HexNAc1Hex1Neu5Ac1 were able to discriminate AD from controls and from patients suffering from other types of dementia. Regarding CSF N-glycosylation, high proportions of high-mannose, biantennary bisecting core-fucosylated N-glycans were found, whereby only about 20% of the N-glycans were sialylated. O-Glycans and free-glycan fragments were less sialylated in AD patients than in controls. To conclude, this comprehensive study revealed for the first time the biomarker potential of free glycans for the differential diagnosis of AD.

Comparison of diagnostic routines for suspected Alzheimer's disease patients in US-American and German primary care.

Aim: Thorough diagnostics are a prerequisite for the optimal treatment of Alzheimer's disease (AD). Biomarker-based diagnostics are standard in academia, data on practitioners' diagnostic workups is scarce. Materials & methods: Surveys in German and US healthcare providers (HCP) were conducted regarding diagnostics in presumed AD patients. A subsample of 153 German and 88 US professionals was analyzed in detail. Results: Fewer German physicians conduct AD diagnostics themselves compared with US colleagues (67% vs 99%; p < 0.0001). German doctors more often order diagnostics at other institutions (65% vs 45%; p < 0.005). No significant differences were found regarding the type of diagnostics ordered at other institutions. Conclusion: Diagnostic routines for suspected AD patients differ between German and US-American healthcare providers.

It is important to conduct the best-possible tests to come to a correct diagnosis of Alzheimer's disease (AD). This ensures choosing the optimal treatment. In academic surroundings such as specialized memory clinics, so called biomarkers (found for example in blood) are an important component in finding the correct diagnosis. However, there is limited data on the methods healthcare providers (HCP) use in their everyday clinical practice. With this study, we aimed to get a clearer picture of the differences in the diagnostic routines for potential AD patients implemented by HCPs in two high-income countries, Germany and the USA. We conducted two surveys in 500 German and 100 US HCPs on their AD-diagnostic routines. A comparable subsample of 153 German and 88 US professionals was analyzed in detail. We found that fewer German physicians conduct AD diagnostics themselves compared with their US­American colleagues (67% vs 99%). The other way around, German doctors more often order diagnostics at other institutions (65% vs 45%). However, there were no significant differences in the type of diagnostic procedures ordered at other institutions. In conclusion, diagnostic routines for suspected AD patients differ between German and US­American healthcare providers, such as biomarker-based diagnostics, which German physicians significantly perform less often.

Influence of preanalytical and analytical factors on the quantification of six regulatory serum proteins.

Background: We analyzed differences in protein concentrations in human blood serum depending on the tube material and the immunoassay platform used. Materials & methods: Blood samples from study participants were collected in glass and polypropylene tubes (n = 292). Serum concentrations of six proteins (BDNF, IGF-1, VEGF-A, TGF-ß1, MCP-1 and IL-18) were assessed by using ELISAs (all biomarkers), as well as a novel fully automated immunoassay platform (all but IGF-1, n = 211). Bland-Altman analyses were conducted to investigate intrasample variability of protein concentrations. Results: Tube comparison resulted in mean biases of between -0.45 and -70.64%. Platform comparison revealed mean biases of between 21.04 and -128.10%. Conclusion: Protein concentrations can vary significantly depending on the types of tube and immunoassay used, with protein-specific differences.

This study investigated the impact of blood tube materials and measuring platforms on protein concentrations in blood samples. We collected blood serum from up to 292 study participants using glass and polypropylene tubes. The concentrations of six proteins were analyzed using a common laboratory technique called ELISA, as well as an automated platform, Ella™. The choice of tube material had small effects on two proteins (IGF-1 and IL-18), with differences of less than 1%. However, the concentrations of four other proteins (VEGF-A, MCP-1, TGF-ß1 and BDNF) varied significantly more depending on the tube material used, with differences ranging from -32.17 to -70.64%. With the two testing methods, two proteins (VEGF-A and TGF-ß1) showed only small differences, with variations of -7.68 and 11.74%, respectively. For the other four proteins, the differences were larger, from 21.04 to -128.10%. The study demonstrates the importance of having consistent, standardized methods for measuring protein levels in blood samples. The tubes and testing methods used can both change the results significantly, depending on the specific protein being measured. To make sure the measurements are accurate, we suggest creating specific guidelines for each testing method and protein. By following these guidelines, scientists can ensure that the measurements of protein levels in liquid biopsy samples are dependable and consistent.

Long-term stability and age-dependence of six regulatory serum proteins.

Aim: The development of biomarker-based diagnostic procedures often relies on samples stored for several years. We aimed to investigate the influence of storage time and patient age on six neuroregulatory and immunoregulatory serum biomarkers. Materials & methods: We quantified six biomarkers in serum from 151 individuals using ELISA. Serum was stored at -80°C for up to 9.5 years. Results: When associating storage time with biomarker values, BDNF, VEGF-A and TGF-ß1 showed a significant increase over time; IGF-1, MCP-1 and IL-18 did not. Associating participant age with biomarkers, only IL-18 in Alzheimer's disease patients showed a significant increase. Conclusion: Storage time can influence results of biomarkers in human serum. This needs to be considered when assessing samples stored for several years.

Presenilin 1 Gene Mutation (M139V) in a German Family with Early-Onset Alzheimer's Disease: A Case Report.

OBJECTIVE: This study describes a 44-year-old German male with early-onset Alzheimer's disease as a result of a M139V presenilin 1 mutation. The patient has at least seven affected family members, spanning at least four generations. METHOD: We performed a complete demographic, genetic, neuropsychological, neuropsychiatric, neuroradiological, and neuropathological characterizations of this patient. The findings were compared with previous reports of patients with the same mutation. Demographic, neuropsychological, neuropsychiatric, neuroradiological, and neuropathological data from several affected members of the patient's family were also addressed. RESULTS: We describe similarities shared with other cases, including age at onset, rapid disease progression, severe deficits in arithmetic and visuo-constructive abilities with relative preservation of naming skills, and the presence of predominant frontal behavioral symptoms. Differences with respect to previously described cases, including the absence of positive neurological or radiological findings, psychotic symptoms, or a depressive disorder, are also identified and discussed. CONCLUSIONS: Heterogeneity in symptoms between affected patients from the same or from different families suggests that individual, genetic, or epigenetic factors most likely modulate the phenotype of patients carrying the M139V mutation.

Diagnose it yourself: will there be a home test kit for Alzheimer's disease?

Alzheimer's disease is the most common neurodegenerative process leading to dementia. To date, there is no curative approach; thus, establishing a diagnosis as early as possible is necessary to implement preventive measures. However, today's gold standard for diagnosing Alzheimer's disease is high in both cost and effort and is not readily available. This defines the need for low-effort and economic alternatives that give patients low-threshold access to testing systems at their general practitioners or even at home for an independent retrieval of a biologic specimen. This perspective gives an overview of established and novel approaches in the field and speculates on the future of test strategies eventually technically implementable at home.

Lay abstract Alzheimer's disease is a common cause for dementia. While there is no cure yet, finding a diagnosis as early as possible is necessary to slow down worsening of cognitive abilities as much as possible. The commonly administered diagnostic tools for Alzheimer's disease are high in both cost and effort. This emphasizes the need for low-effort and economic alternatives, that give patients a low-threshold access to testing systems at their general practitioners or at home in a self-application. This perspective gives an overview of today's diagnostic standard and reviews novel approaches in the field. It also speculates on the future of strategies that might potentially be suitable for taking a diagnostic test at home.

Value of Neuropsychological Tests to Identify Patients with Depressive Symptoms on the Alzheimer's Disease Continuum.

BACKGROUND: Depressive symptoms often co-occur with Alzheimer's disease (AD) and can impact neuropsychological test results. In early stages of AD, disentangling cognitive impairments due to depression from those due to neurodegeneration often poses a challenge. OBJECTIVE: We aimed to identify neuropsychological tests able to detect AD-typical pathology while taking into account varying degrees of depressive symptoms. METHODS: A battery of neuropsychological tests (CERAD-NP) and the Geriatric Depression Scale (GDS) were assessed, and cerebrospinal fluid (CSF) biomarkers were obtained. After stratifying patients into CSF positive or negative and into low, moderate, or high GDS score groups, sensitivity and specificity and area under the curve (AUC) were calculated for each subtest. RESULTS: 497 participants were included in the analyses. In patients with low GDS scores (≤10), the highest AUC (0.72) was achieved by Mini-Mental State Examination, followed by Constructional Praxis Recall and Wordlist Total Recall (AUC = 0.714, both). In patients with moderate (11-20) and high (≥21) GDS scores, Trail Making Test-B (TMT-B) revealed the highest AUCs with 0.77 and 0.82, respectively. CONCLUSION: Neuropsychological tests showing AD-typical pathology in participants with low GDS scores are in-line with previous results. In patients with higher GDS scores, TMT-B showed the best discrimination. This indicates the need to focus on executive function rather than on memory task results in depressed patients to explore a risk for AD.

Plasma Amyloid Concentration in Alzheimer's Disease: Performance of a High-Throughput Amyloid Assay in Distinguishing Alzheimer's Disease Cases from Controls.

BACKGROUND: Collection of cerebrospinal fluid (CSF) for measurement of amyloid-ß (Aß) species is a gold standard in Alzheimer's disease (AD) diagnosis, but has risks. Thus, establishing a low-risk blood Aß test with high AD sensitivity and specificity is of outmost interest. OBJECTIVE: We evaluated the ability of a commercially available plasma Aß assay to distinguish AD patients from biomarker-healthy controls. METHOD: In a case-control design, we examined plasma samples from 44 AD patients (A + N+) and 49 controls (A-N-) from a memory clinic. AD was diagnosed using a combination of neuropsychological examination, CSF biomarker analysis and brain imaging. Total Aß40 and total Aß42 in plasma were measured through enzyme-linked immunosorbent assay (ELISA) technology using ABtest40 and ABtest42 test kits (Araclon Biotech Ltd.). Receiver operating characteristic (ROC) analyses with outcome AD were performed, and sensitivity and specificity were calculated. RESULTS: Plasma Aß42/40 was weakly positively correlated with CSF Aß42/40 (Spearman's rho 0.22; p = 0.037). Plasma Aß42/40 alone was not able to statistically significantly distinguish between AD patients and controls (AUC 0.58; 95% CI 0.46, 0.70). At a cut-point of 0.076 maximizing sensitivity and specificity, plasma Aß42/40 had a sensitivity of 61.2% and a specificity of 63.6%. CONCLUSION: In this sample, the high-throughput blood Aß assay was not able to distinguish well between AD patients and controls. Whether or not the assay may be useful in large-scale epidemiological settings remains to be seen.

Pursuing Experimental Reproducibility: An Efficient Protocol for the Preparation of Cerebrospinal Fluid Samples for NMR-based Metabolomics and Analysis of Sample Degradation.

NMR-based metabolomics investigations of human biofluids offer great potential to uncover new biomarkers. In contrast to protocols for sample collection and biobanking, procedures for sample preparation prior to NMR measurements are still heterogeneous, thus compromising the comparability of the resulting data. Herein, we present results of an investigation of the handling of cerebrospinal fluid (CSF) samples for NMR metabolomics research. Origins of commonly observed problems when conducting NMR experiments on this type of sample are addressed, and suitable experimental conditions in terms of sample preparation and pH control are discussed. Sample stability was assessed by monitoring the degradation of CSF samples by NMR, hereby identifying metabolite candidates, which are potentially affected by sample storage. A protocol was devised yielding consistent spectroscopic data as well as achieving overall sample stability for robust analysis. We present easy to adopt standard operating procedures with the aim to establish a shared sample handling strategy that facilitates and promotes inter-laboratory comparison, and the analysis of sample degradation provides new insights into sample stability.

Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors.

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer's disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD.

Astrocytes discriminate and selectively respond to the activity of a subpopulation of neurons within the barrel cortex.

Sensory information from single whiskers in rodents projects to defined morphological units in the cortex, the barrels. We found that astrocytes selectively respond with an increase in the cytosolic Ca(2+) concentration to activation of layer 4 neurons, the input cells of the barrel columns. The neuronal Ca(2+) signal also spread across barrel column borders mainly in layer 2/3, but the glutamate-mediated astrocyte response stayed restricted to the barrel column. In contrast, when interfering with inhibitory pathways by blocking either purinergic, adenosine or gamma-aminobutyric acid(A) receptors, the stimulation activated a Ca(2+) response in a much larger astrocyte population no longer restricted to the borders of the barrel column. We also observed spontaneous and evoked Ca(2+) activity in the synaptic target cells of layer 4 neurons, the layer 2/3 pyramidal cells, but again, we never recorded Ca(2+) responses in astrocytes following activity in this neuronal population. Our data show that astrocytes can discriminate and selectively respond to the activity of a subpopulation of excitatory neurons within a given brain region. This selectivity in the astrocyte response describes a new level of complexity and integration in the reaction of astrocytes to neuronal activity.

Definition and quantification of six immune- and neuroregulatory serum proteins in healthy and demented elderly.

Aim: Blood-based biomarkers related to immune- and neuroregulatory processes may be indicative of dementia but lack standardization and proof-of-principle studies. Materials & methods: The blood serum collection protocol as well as the analytic procedure to quantify the markers BDNF, IGF-1, VEGF, TGF-ß 1, MCP-1 and IL-18 in blood serum were standardized and their concentrations were compared between groups of 81 Alzheimer's disease patients and 79 healthy controls. Results: Applying standardized methods, results for the quantification of the six markers in blood serum are stable and their concentrations significantly differ for all analytes except VEGF between patients diagnosed with Alzheimer's disease and healthy controls. Conclusion: Analyzing a panel of six markers in blood serum under standardized conditions may serve as a diagnostic tool in primary dementia care in the future.

Temperature and nitric oxide control spontaneous calcium transients in astrocytes.

Transient spontaneous increases in the intracellular Ca2+ concentration have been frequently observed in astrocytes in cell culture and in acutely isolated slices from several brain regions. Recent in vivo experiments, however, reported only a low frequency of spontaneous Ca2+ events in astrocytes. Since the ex vivo experiments were usually performed at temperatures lower than physiological body temperature, we addressed the question whether temperature could influence the spontaneous Ca2+ activity in astrocytes. Indeed, comparing the frequency and spike width of spontaneous Ca2+ transients in astrocytes at temperatures between 20 and 37 degrees C in culture as well as in acute cortical slices from mouse brain, revealed that spontaneous Ca2+ responses occurred frequently at low temperature and became less frequent at higher temperature. Moreover, the single Ca2+ events had a longer duration at low temperature. We found that nitric oxide (NO) mimicked the increase in spontaneous Ca2+ activity and that an NO-synthase inhibitor attenuated the effect of lowering the temperature. Thus, temperature and NO are major determinants of spontaneous astrocytic Ca2+ signalling.

Methods in endogenous steroid profiling - A comparison of gas chromatography mass spectrometry (GC-MS) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS).

In various fields of endocrinology, the determination of steroid hormones synthesised by the human body plays an important role. Research on central neurosteroids has been intensified within the last years, as they are discussed as biomarkers for various cognitive disorders. Their concentrations in cerebrospinal fluid (CSF) are considered to be regulated independently from peripheral fluids. For that reason, the challenging matrix CSF becomes a very interesting specimen for analysis. Concentrations are expected to be very low and available amount of CSF is limited. Thus, a comprehensive method for very sensitive quantification of a set of analytes as large as possible in one analytical aliquot is desired. However, high structural similarities of the selected panel of 51 steroids and steroid sulfates, including numerous isomers, challenges achievement of chromatographic selectivity. Since decades the analysis of endogenous steroids in various body fluids is mainly performed by gas chromatography (GC) coupled to (tandem) mass spectrometry (MS(/MS)). Due to the structure of the steroids of interest, derivatisation is performed to meet the analytical requirements for GC-MS(/MS). Most of the laboratories use a two-step derivatisation in multi-analyte assays that was already published in the 1980s. However, for some steroids this elaborate procedure yields multiple isomeric derivatives. Thus, some laboratories utilize (ultra) high performance liquid chromatography ((U)HPLC)-MS/MS as alternative but, even UHPLC is not able to separate some of the isomeric pairs. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to GC and (U)HPLC may help to overcome these issues. Within this project the two most promising methods for endogenous steroid profiling were investigated and compared: the "gold standard" GC-MS and the orthogonal separation technique SFC-MS/MS. Different derivatisation procedures for gas chromatographic detection were explored and the formation of multiple derivatives described and confirmed. Taken together, none of the investigated derivatisation procedures provided acceptable results for further method development to meet the requirements of this project. SFC with its unique selectivity was able to overcome these issues and to distinguish all selected steroids, including (pro-)gestagens, androgens, corticoids, estrogens, and steroid sulfates with appropriate selectivity. Valued especially in the separation of enantiomeric analytes, SFC has shown its potential as alternative to GC. The successful separation of 51 steroids and steroid sulfates on different columns is presented to demonstrate the potential of SFC in endogenous steroid profiling.

Neurogranin and BACE1 in CSF as Potential Biomarkers Differentiating Depression with Cognitive Deficits from Early Alzheimer's Disease: A Pilot Study.

BACKGROUND/AIMS: Major depressive disorder (MDD) can cooccur with early Alzheimer's disease (AD) or may cause memory problems independently of AD. Previous studies have suggested that the AD-related cerebrospinal fluid (CSF) biomarkers tau and Aß(1-42) could help discriminate between early AD and depression unrelated to AD. Moreover, the postsynaptic protein neurogranin and presynaptic BACE1 have increasingly gained attention as potential new AD biomarkers, but they have not yet been investigated concerning depression. METHODS: Using ELISAs, we studied CSF neurogranin and BACE1 levels in patients with mild (n = 21) and moderate (n = 19) AD, as well as in MDD patients with (n = 20) and without (n = 20) cognitive deficits. The clinical examinations included analyses of t-tau, Aß(1-42), and Aß(1-40), besides neuropsychological tests and cranial magnetic resonance imaging. Depressive symptom severity was assessed using the Geriatric Depression Scale (GDS). RESULTS: Along with classic AD biomarkers, neurogranin and BACE1 CSF levels differed between moderate AD and MDD (p ≤ 0.01). MDD associated with cognitive deficits was distinguished from mild AD through the CSF neurogranin/BACE1 ratio (p < 0.05), which was strongly correlated with GDS scores (ρ = -0.656; p < 0.01). CONCLUSION: The neurogranin/BACE1 ratio in CSF can distinguish between depression and AD among patients with similar cognitive deficits, along with the classic AD biomarkers. Further longitudinal studies are ongoing to identify which biomarkers have prognostic value.

Distinct expression of the neurotoxic microRNA family let-7 in the cerebrospinal fluid of patients with Alzheimer's disease.

MicroRNAs (miRNAs) are non-coding RNAs originally involved in RNA silencing and post-transcriptional regulation of gene expression. We have shown in previous work that the miRNA let-7b can act as a signalling molecule for Toll-like receptor 7, thereby initiating innate immune pathways and apoptosis in the central nervous system. Here, we investigated whether different members of the miRNA family let-7, abundantly expressed in the brain, are released into the human cerebrospinal fluid (CSF) and whether quantitative differences in let-7 copies exist in neurodegenerative diseases. RNA isolated from CSF of patients with Alzheimer´s disease (AD) and from control patients with frontotemporal lobe dementia (FTLD), major depressive episode (MDE) without clinical or neurobiological signs of AD, and healthy individuals, was reverse transcribed with primers against nine let-7 family members, and miRNAs were quantified and analyzed comparatively by quantitative PCR. let-7 miRNAs were present in CSF from patients with AD, FTLD, MDE, and healthy controls. However, the amount of individual let-7 miRNAs in the CSF varied substantially. CSF from AD patients contained higher amounts of let-7b and let-7e compared to healthy controls, while no differences were observed regarding the other let-7 miRNAs. No increase in let-7b and let-7e was detected in CSF from FTLD patients, while in CSF from MDE patients, let-7b and let-7e copy levels were elevated. In CSF from AD patients, let-7b and let-7e were associated with extracellular vesicles. let-7 family members present in the CSF mediated neurotoxicity in vitro, albeit to a variable extent. Taken together, neurotoxic let-7 miRNAs are differentially and specifically released in AD, but also in MDE patients. Thus, these miRNAs may mirror common neuropathological paths and by this serve to unscramble mechanisms of different neurodegenerative diseases.

Golgin A4 in CSF and granulovacuolar degenerations of patients with Alzheimer disease.

OBJECTIVE: To isolate and identify a new, as yet unknown molecule in CSF that could serve as marker for Alzheimer disease. METHODS: We immunized mice with human CSF and fused hybridomas for monoclonal antibodies and screened these antibodies for their capacity to discriminate CSF of patients with Alzheimer disease from CSF of controls. We then chromatographically isolated the antigen to the best discriminating antibody and identified the antigen using mass spectrometric methods. Thereafter, we quantified the CSF concentration of the antigen in a new cohort of patients with Alzheimer disease and controls and performed immunohistochemistry of postmortem brain tissue derived from patients with Alzheimer disease and controls. RESULTS: We generated >200 hybridomas and selected 1 antibody that discriminated CSF from patients with Alzheimer disease from that of controls. We identified golgin A4 as the antigen detected by this antibody. Golgin A4 concentration was significantly higher in CSF from patients with Alzheimer disease than in CSF of controls (145 [interquartile range 125-155] vs 115 [ 99-128] pg/mL, p < 0.001) and demonstrated a substantial discriminative power (area under the receiver operating characteristic curve 0.80, 95% confidence interval 0.67-0.94). Immunohistochemistry of postmortem brain sections from patients with Alzheimer disease revealed a significant accumulation of golgin A4 in granulovacuolar degeneration bodies (GVBs). CONCLUSIONS: These results support the notion that golgin A4 could serve as a diagnostic marker in Alzheimer disease. For validation of this notion, prospective multicenter diagnostic studies will evaluate golgin A4 as diagnostic marker for Alzheimer disease. Furthermore, it has to be determined whether the association of golgin A4 with GVBs is an epiphenomenon or whether golgin A4 plays a more direct role in Alzheimer disease, allowing it to serve as a target in therapeutic treatment strategies. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that elevated CSF golgin A4 levels identify patients with Alzheimer disease.