Impaired sleep is a common aspect of aging and often precedes the onset of Alzheimer’s disease. Here, we compare the effects of sleep deprivation in young wild-type mice and their APP/PS1 littermates, a murine model of Alzheimer’s disease. After 7 h of sleep deprivation, both genotypes exhibit an increase in EEG slow-wave activity. However, only the wild-type mice demonstrate an increase in the power of infraslow norepinephrine oscillations, which are characteristic of healthy non-rapid eye movement sleep. Notably, the APP/PS1 mice fail to enhance norepinephrine oscillations 24 h after sleep deprivation, coinciding with an accumulation of cerebral amyloid-β protein. Proteome analysis of cerebrospinal fluid and extracellular fluid further supports these findings by showing altered protein clearance in APP/PS1 mice. We propose that the suppression of infraslow norepinephrine oscillations following sleep deprivation contributes to increased vulnerability to sleep loss and heightens the risk of developing amyloid pathology in early stages of Alzheimer’s disease.

AbstractDisease-modifying therapies for Alzheimer’s disease (AD) are likely to be most beneficial when initiated in the presymptomatic phase. To track the benefit of such interventions, fluid biomarkers are of great importance, with neurofilament light chain protein (NfL) showing promise for monitoring neurodegeneration and predicting cognitive outcomes. Here, we update and complement previous findings from the Dominantly Inherited Alzheimer Network Observational Study by using matched cross-sectional and longitudinal cerebrospinal fluid (CSF) and plasma samples from 567 individuals, allowing timely comparative analyses of CSF and blood trajectories across the entire disease spectrum. CSF and plasma trajectories were similar at presymptomatic stages, discriminating mutation carriers from non-carrier controls 10-20 years before the estimated onset of clinical symptoms, depending on the statistical model used. However, after symptom onset the rate of change in CSF NfL continued to increase steadily, whereas the rate of change in plasma NfL leveled off. Both plasma and CSF NfL changes were associated with grey-matter atrophy, but not with Aβ-PET changes, supporting a temporal decoupling of Aβ deposition and neurodegeneration. These observations support NfL in both CSF and blood as an early marker of neurodegeneration but suggest that NfL measured in the CSF may be better suited for monitoring clinical trial outcomes in symptomatic AD patients.

Human induced pluripotent stem cells (iPSCs) have transpired as an attractive tool to investigate neurological diseases. And while immune cells, and in particular microglia, have emerged as important players in many neurological and in particular in neurodegenerative diseases, it remains a challenge to fully recapitulate an in vivo-like phenotype ex vivo. This is due to the fact that microglial identity is shaped by the neural tissue environment. We have now developed a chimeric brain slice culture system, where we engraft iPSC-derived microglia onto mouse (and human) organotypic brain slice cultures. Brain slice cultures offer the advantage that many aspects of the complex cytoarchitecture of the mature/aged living brain, including cortical layering, columnar organization and electrophysiological properties can be preserved over several weeks. We found that iPSC-derived microglial precursors integrate and differentiate well in the brain slice cultures, with morphology, network characteristics and functional responses reminiscent of human microglia. Gene expression profiling of iPSC-derived microglia revealed enhanced maturation over time. Upon induction of neurodegenerative disease pathology in these slice cultures, the iPSC-derived microglia show transcriptional changes converging towards those observed in patients with neurodegenerative disease pathology. This system is a novel cellularly complex tool allowing to dissect the dynamics and molecular mechanisms of microglial genetic risk factors of many neurological diseases, while preserving experimental amenability to test therapeutic approaches in a human(ized) system.

Significance Single-cell transcriptomics has revealed specific glial activation states associated with the pathogenesis of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease (AD and PD). What is still needed are clinically relevant biomarkers for deciphering such glial states in AD and PD patients. To this end, we applied proteome analysis in cerebrospinal fluid (CSF) of mouse models of AD and PD pathology. This allowed us to identify a panel of glial CSF proteins that largely match the transcriptomic changes. The identified proteins can also be quantified in human CSF and show changes in AD patients, supporting their relevance as biomarker candidates to stage glial activation in patients with neurodegenerative diseases.

Brain Aβ deposition is a key early event in the pathogenesis of Alzheimer´s disease (AD), but the long presymptomatic phase and poor correlation between Aβ deposition and clinical symptoms remain puzzling. To elucidate the dependency of downstream pathologies on Aβ, we analyzed the trajectories of cerebral Aβ accumulation, Aβ seeding activity, and neurofilament light chain (NfL) in the CSF (a biomarker of neurodegeneration) in Aβ-precursor protein transgenic mice. We find that Aβ deposition increases linearly until it reaches an apparent plateau at a late age, while Aβ seeding activity increases more rapidly and reaches a plateau earlier, coinciding with the onset of a robust increase of CSF NfL. Short-term inhibition of Aβ generation in amyloid-laden mice reduced Aβ deposition and associated glial changes, but failed to reduce Aβ seeding activity, and CSF NfL continued to increase although at a slower pace. When short-term or long-term inhibition of Aβ generation was started at pre-amyloid stages, CSF NfL did not increase despite some Aβ deposition, microglial activation, and robust brain Aβ seeding activity. A dissociation of Aβ load and CSF NfL trajectories was also found in familial AD, consistent with the view that Aβ aggregation is not kinetically coupled to neurotoxicity. Rather, neurodegeneration starts when Aβ seeding activity is saturated and before Aβ deposition reaches critical (half-maximal) levels, a phenomenon reminiscent of the two pathogenic phases in prion disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-β precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-β deposits, and that in mice, medin deficiency reduces vascular amyloid-β deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-β burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-β to promote its aggregation, as medin forms heterologous fibrils with amyloid-β, affects amyloid-β fibril structure, and cross-seeds amyloid-β aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-β deposition in the blood vessels of the brain.

There is a large gap between the growing awareness regarding the importance of microglia in the pathogenesis of neurodegenerative disorders like Alzheimer's and Parkinson's disease and the availability of microglia-related molecular biomarkers in practice. Heterogeneity due to comorbidities in human cohorts together with cost-intensive technologies is a major challenge in the search for novel CSF and blood-borne biomarkers. Transgenic mouse models, however, become increasingly appreciated for the interpretation of fluid biomarker changes due to low variability and well-defined pathomechanisms. We applied a label-free LC-MS/MS approach in order to do shotgun proteomics in CSF samples from two well-characterized mouse models of beta-amyloidosis and alpha-synucleinopathy, APPPS1 (Radde et al. 2006 EMBO Rep) and A30PaS (Neumann et al. 2002 J Clin Invest), respectively. CSF was collected from 3- and 18-month-old APPPS1 and 3-, 11- and 18-month-old A30PaS and aged-matched non-transgenic mice (n=5–10 per group) to analyze age- and transgene-related protein changes. A subset of findings was subsequently validated by Western blot or sandwich-immunoassay. Our proteome screen identified 1,168 and 1,261 unique proteins in the CSF of APPPS1 and A30PaS mice, respectively. Label free quantification revealed no protein changes in young mice. However, eight proteins were significantly increased in aged APPPS1 mice after FDR correction, whereas 220 proteins showed a difference (predominantly increase) in 18-month-old A30PaS compared to non-transgenic control mice. Strikingly, roughly half of the 24 top hits showing an age- and transgene-related increase in both mouse models (p<0.05) were linked to microglia. Moreover, eight of these proteins have recently been assigned to a specific disease-associated microglia (DAM) phenotype (Keren-Shaul et al. 2017 Cell). Among them TREM2, a previously identified CSF biomarker and key player during DAM transition from activation stage 1 to 2. The current mouse CSF proteome study reveals a novel panel of microglia-related analytes including soluble TREM2. Their robust age-related increase, observed in two mouse models of distinct neurodegenerative diseases, holds promise in the search for much awaited reliable biomarkers to track the complex immune response to CNS damage in humans.

A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases.

Abnormalities in brains of Alzheimer's disease (AD) patients are thought to start long before the first clinical symptoms emerge. The identification of affected individuals at this 'preclinical AD' stage relies on biomarkers such as decreased levels of the amyloid-β peptide (Aβ) in the cerebrospinal fluid (CSF) and positive amyloid positron emission tomography scans. However, there is little information on the longitudinal dynamics of CSF biomarkers, especially in the earliest disease stages when therapeutic interventions are likely most effective. To this end, we have studied CSF Aβ changes in three Aβ precursor protein transgenic mouse models, focusing our analysis on the initial Aβ deposition, which differs significantly among the models studied. Remarkably, while we confirmed the CSF Aβ decrease during the extended course of brain Aβ deposition, a 20-30% increase in CSF Aβ40 and Aβ42 was found around the time of the first Aβ plaque appearance in all models. The biphasic nature of this observed biomarker changes stresses the need for longitudinal biomarker studies in the clinical setting and the search for new 'preclinical AD' biomarkers at even earlier disease stages, by using both mice and human samples. Ultimately, our findings may open new perspectives in identifying subjects at risk for AD significantly earlier, and in improving the stratification of patients for preventive treatment strategies.

Alzheimer's Disease (AD) is thought to start 10–20 years before the first clinical symptoms emerge. Notably, familial AD patients already show decreased CSF Aβ42 and increasedtotal-Tau (t-Tau) levels in this pre-clinical phase. It is assumed that the drop in CSF Aβ42 is associated with Aβ aggregation and deposition in the brain parenchyma and that CSF t-Tau increase reflects the extracellular release of Tau after neuronal degeneration. These interpretations are largely indirect, based on CSF measurements during life vs. neuropathological assessment at autopsy, and on analogies with episodic CNS conditions (e.g. stroke, head trauma). To overcome these limitations, APP transgenic (APP-tg) mice may represent a reliable model to characterize AD-related CSF biomarker dynamics. We analyzed CSF biomarker profiles in a large series of well-characterized APP-tg mice: APPPS1 and APP23, virtually covering all steps from pre-depositing to late depositing stages. CSF and brain human- A β40, A β42 and endogenous t-Tau measurements were performed by Electrochemiluminescence-linked immunoassay. Plaque load was determined by stereology on histological sections. In APP23 mice CSF A β42 levels remained stable with a slight increase before brain Aβ deposition started, which was then followed by a significant drop when A βdeposition became prominent. This was further reinforced in APPPS1 mice, an early A β depositing model, where CSF human A β42 levels exhibited an immediate and striking decline, reaching 80% in aged mice. CSF t-Tau increased remarkably following A β deposition in both mouse models. The CSF t-Tau increase occurred earlier and more markedly in APPPS1, compared to APP23 mice, despite the higher APP expression in the latter mouse model. Overall, the temporal sequence of the CSF biomarker changes in both APP-tg was characterized by a decline in CSF A β42 with increasing brain A β deposition, which was followed by a steady CSF t-Tau increase, in the absence of neurofibrillary tangles and significant neuronal loss. We have shown that the CSF Aβ decrease reflects brain A β-amyloidosis and that Aβ aggregation in the brain induces CSF tau increase in the absence of neurofibrillary tangles and major neuron loss. This temporal profile of CSF biomarkers makes APP-tg mice invaluable translational tools for preclinical prevention and disease-modifying therapies.