Organotypic Hippocampal Slice Cultures from Adult Tauopathy Mice and Theragnostic Evaluation of Nanomaterial Phospho-TAU Antibody-Conjugates.

Organotypic slice culture models surpass conventional in vitro methods in many aspects. They retain all tissue-resident cell types and tissue hierarchy. For studying multifactorial neurodegenerative diseases such as tauopathies, it is crucial to maintain cellular crosstalk in an accessible model system. Organotypic slice cultures from postnatal tissue are an established research tool, but adult tissue-originating systems are missing, yet necessary, as young tissue-originating systems cannot fully model adult or senescent brains. To establish an adult-originating slice culture system for tauopathy studies, we made hippocampal slice cultures from transgenic 5-month-old hTau.P301S mice. In addition to the comprehensive characterization, we set out to test a novel antibody for hyperphosphorylated TAU (pTAU, B6), with and without a nanomaterial conjugate. Adult hippocampal slices retained intact hippocampal layers, astrocytes, and functional microglia during culturing. The P301S-slice neurons expressed pTAU throughout the granular cell layer and secreted pTAU to the culture medium, whereas the wildtype slices did not. Additionally, cytotoxicity and inflammation-related determinants were increased in the P301S slices. Using fluorescence microscopy, we showed target engagement of the B6 antibody to pTAU-expressing neurons and a subtle but consistent decrease in intracellular pTAU with the B6 treatment. Collectively, this tauopathy slice culture model enables measuring the extracellular and intracellular effects of different mechanistic or therapeutic manipulations on TAU pathology in adult tissue without the hindrance of the blood-brain barrier.

Protective Alzheimer's disease-associated APP A673T variant predominantly decreases sAPPß levels in cerebrospinal fluid and 2D/3D cell culture models.

The rare A673T variant was the first variant found within the amyloid precursor protein (APP) gene conferring protection against Alzheimer's disease (AD). Thereafter, different studies have discovered that the carriers of the APP A673T variant show reduced levels of amyloid beta (Aß) in the plasma and better cognitive performance at high age. Here, we analyzed cerebrospinal fluid (CSF) and plasma of APP A673T carriers and control individuals using a mass spectrometry-based proteomics approach to identify differentially regulated targets in an unbiased manner. Furthermore, the APP A673T variant was introduced into 2D and 3D neuronal cell culture models together with the pathogenic APP Swedish and London mutations. Consequently, we now report for the first time the protective effects of the APP A673T variant against AD-related alterations in the CSF, plasma, and brain biopsy samples from the frontal cortex. The CSF levels of soluble APPß (sAPPß) and Aß42 were significantly decreased on average 9-26% among three APP A673T carriers as compared to three well-matched controls not carrying the protective variant. Consistent with these CSF findings, immunohistochemical assessment of cortical biopsy samples from the same APP A673T carriers did not reveal Aß, phospho-tau, or p62 pathologies. We identified differentially regulated targets involved in protein phosphorylation, inflammation, and mitochondrial function in the CSF and plasma samples of APP A673T carriers. Some of the identified targets showed inverse levels in AD brain tissue with respect to increased AD-associated neurofibrillary pathology. In 2D and 3D neuronal cell culture models expressing APP with the Swedish and London mutations, the introduction of the APP A673T variant resulted in lower sAPPß levels. Concomitantly, the levels of sAPPα were increased, while decreased levels of CTFß and Aß42 were detected in some of these models. Our findings emphasize the important role of APP-derived peptides in the pathogenesis of AD and demonstrate the effectiveness of the protective APP A673T variant to shift APP processing towards the non-amyloidogenic pathway in vitro even in the presence of two pathogenic mutations.

MECP2 Increases the Pro-Inflammatory Response of Microglial Cells and Phosphorylation at Serine 423 Regulates Neuronal Gene Expression upon Neuroinflammation.

Methyl-CpG-binding protein 2 (MECP2) is a critical transcriptional regulator for synaptic function. Dysfunction of synapses, as well as microglia-mediated neuroinflammation, represent the earliest pathological events in Alzheimer's disease (AD). Here, expression, protein levels, and activity-related phosphorylation changes of MECP2 were analyzed in post-mortem human temporal cortex. The effects of wild type and phosphorylation-deficient MECP2 variants at serine 423 (S423) or S80 on microglial and neuronal function were assessed utilizing BV2 microglial monocultures and co-cultures with mouse cortical neurons under inflammatory stress conditions. MECP2 phosphorylation at the functionally relevant S423 site nominally decreased in the early stages of AD-related neurofibrillary pathology in the human temporal cortex. Overexpression of wild type MECP2 enhanced the pro-inflammatory response in BV2 cells upon treatment with lipopolysaccharide (LPS) and interferon-γ (IFNγ) and decreased BV2 cell phagocytic activity. The expression of the phosphorylation-deficient MECP2-S423A variant, but not S80A, further increased the pro-inflammatory response of BV2 cells. In neurons co-cultured with BV2 cells, the MECP2-S423A variant increased the expression of several genes, which are important for the maintenance and protection of neurons and synapses upon inflammatory stress. Collectively, functional analyses in different cellular models suggest that MECP2 may influence the inflammatory response in microglia independently of S423 and S80 phosphorylation, while the S423 phosphorylation might play a role in the activation of neuronal gene expression, which conveys neuroprotection under neuroinflammation-related stress.

Diabetic phenotype in mouse and humans reduces the number of microglia around ß-amyloid plaques.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease and type 2 diabetes (T2D) plays an important role in conferring the risk for AD. Although AD and T2D share common features, the common molecular mechanisms underlying these two diseases remain elusive. METHODS: Mice with different AD- and/or tauopathy-linked genetic backgrounds (APPswe/PS1dE9, Tau P301L and APPswe/PS1dE9/Tau P301L) were fed for 6 months with standard diet or typical Western diet (TWD). After behavioral and metabolic assessments of the mice, the effects of TWD on global gene expression as well as dystrophic neurite and microglia pathology were elucidated. Consequently, mechanistic aspects related to autophagy, cell survival, phagocytic uptake as well as Trem2/Dap12 signaling pathway, were assessed in microglia upon modulation of PI3K-Akt signaling. To evaluate whether the mouse model-derived results translate to human patients, the effects of diabetic phenotype on microglial pathology were assessed in cortical biopsies of idiopathic normal pressure hydrocephalus (iNPH) patients encompassing ß-amyloid pathology. RESULTS: TWD led to obesity and diabetic phenotype in all mice regardless of the genetic background. TWD also exacerbated memory and learning impairment in APPswe/PS1dE9 and Tau P301L mice. Gene co-expression network analysis revealed impaired microglial responses to AD-related pathologies in APPswe/PS1dE9 and APPswe/PS1dE9/Tau P301L mice upon TWD, pointing specifically towards aberrant microglial functionality due to altered downstream signaling of Trem2 and PI3K-Akt. Accordingly, fewer microglia, which did not show morphological changes, and increased number of dystrophic neurites around ß-amyloid plaques were discovered in the hippocampus of TWD mice. Mechanistic studies in mouse microglia revealed that interference of PI3K-Akt signaling significantly decreased phagocytic uptake and proinflammatory response. Moreover, increased activity of Syk-kinase upon ligand-induced activation of Trem2/Dap12 signaling was detected. Finally, characterization of microglial pathology in cortical biopsies of iNPH patients revealed a significant decrease in the number of microglia per ß-amyloid plaque in obese individuals with concomitant T2D as compared to both normal weight and obese individuals without T2D. CONCLUSIONS: Collectively, these results suggest that diabetic phenotype in mice and humans mechanistically associates with abnormally reduced microglial responses to ß-amyloid pathology and further suggest that AD and T2D share overlapping pathomechanisms, likely involving altered immune function in the brain.

The Alzheimer's disease-associated protective Plcγ2-P522R variant promotes immune functions.

BACKGROUND: Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer's disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD. METHODS: To assess the role of the protective variant in the context of immune cell functions, we generated a Plcγ2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. RESULTS: Functional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plcγ2 as a Pip2-metabolizing enzyme. This was associated with improved survival and increased acute inflammatory response of the KI macrophages. Enhanced phagocytosis was observed in mouse BV2 microglia-like cells overexpressing human PLCγ2-P522R, but not in PLCγ2-WT expressing cells. Immunohistochemical analyses did not reveal changes in the number or morphology of microglia in the cortex of Plcγ2-P522R KI mice. However, the brain mRNA signature together with microglia-related PET imaging suggested enhanced microglial functions in Plcγ2-P522R KI mice. CONCLUSION: The AD-associated protective Plcγ2-P522R variant promotes protective functions associated with TREM2 signaling. Our findings provide further support for the idea that pharmacological modulation of microglia via TREM2-PLCγ2 pathway-dependent stimulation may be a novel therapeutic option for the treatment of AD.

Altered Insulin Signaling in Alzheimer's Disease Brain - Special Emphasis on PI3K-Akt Pathway.

Alzheimer's disease (AD) and type 2 diabetes (T2D) are both diseases with increasing prevalence in aging populations. T2D, characterized by insulin resistance and defective insulin signaling, is a common co-morbidity and a risk factor for AD, increasing the risk approximately two to fourfold. Insulin exerts a wide variety of effects as a growth factor as well as by regulating glucose, fatty acid, and protein metabolism. Certain lifestyle factors, physical inactivity and typical Western diet (TWD) containing high fat and high sugar are strongly associated with insulin resistance and T2D. The PI3K-Akt signaling pathway is a major mediator of effects of insulin and plays a crucial role in T2D pathogenesis. Decreased levels of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) subunits as well as blunted Akt kinase phosphorylation have been observed in the AD brain, characterized by amyloid-ß and tau pathologies. Furthermore, AD mouse models fed with TWD have shown to display altered levels of PI3K subunits. How impaired insulin-PI3K-Akt signaling in peripheral tissues or in the central nervous system (CNS) affects the development or progression of AD is currently poorly understood. Interestingly, enhancement of PI3K-Akt signaling in the CNS by intranasal insulin (IN) treatment has been shown to improve memory in vivo in mice and in human trials. Insulin is known to augment neuronal growth and synapse formation through the PI3K-Akt signaling pathway. However, PI3K-Akt pathway mediates signaling related to different functions also in other cell types, like microglia and astrocytes. In this review, we will discuss the most prominent molecular mechanisms related to the PI3K-Akt pathway in AD and how T2D and altered insulin signaling may affect the pathogenesis of AD.

Decreased plasma C-reactive protein levels in APOE ε4 allele carriers.

OBJECTIVE: Apolipoprotein E (APOE) ε4 allele is a well-established risk factor in Alzheimer's disease (AD). Here, we assessed the effects of APOE polymorphism on cardiovascular, metabolic, and inflammation-related parameters in population-based cohorts. METHODS: Association of cardiovascular, metabolic, and inflammation-related parameters with the APOE polymorphism in a large Finnish Metabolic Syndrome in Men (METSIM) cohort and Finnish Geriatric Intervention study to prevent cognitive impairment and disability (FINGER) were investigated. Brain-specific effects were addressed in postmortem brain samples. RESULTS: Individuals carrying the APOE ε4 allele displayed significantly elevated serum/plasma LDL cholesterol and apolipoprotein B levels. APOE ε3ε4 and ε4ε4 significantly associated with lower levels of plasma high-sensitivity C-reactive protein (hs-CRP). Plasma amyloid-ß 42 (Aß42) and reduced hs-CRP levels showed an association independently of the APOE status. INTERPRETATION: These data suggest that the APOE ε4 allele associates with lower levels of hs-CRP in individuals without dementia. Moreover, Aß42 may encompass anti-inflammatory effects reflected by reduced hs-CRP levels.

DHCR24 exerts neuroprotection upon inflammation-induced neuronal death.

BACKGROUND: DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer's disease. METHODS: Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. RESULTS: Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. CONCLUSIONS: These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo.

Decreased plasma ß-amyloid in the Alzheimer's disease APP A673T variant carriers.

We investigated the association of Alzheimer's disease (AD)-related rare variants APP A673T and ABCA7 rs200538373-C with the levels of ß-amyloid (Aß) and parameters of metabolic and cardiovascular health in a population-based cohort of healthy middle-aged and elderly men. Carriers of protective APP A673T variant had, on average, 28% lower levels of Aß40 and Aß42 in plasma as compared to the controls and the carriers of ABCA7 rs200538373-C. This is the first report to show decreased Aß levels in plasma in APP A673T carriers and thus provides evidence that lower Aß levels throughout life may be protective against AD. Ann Neurol 2017;82:128-132.

Effects of Alzheimer's disease-associated risk loci on cerebrospinal fluid biomarkers and disease progression: a polygenic risk score approach.

BACKGROUND: Several risk loci for Alzheimer's disease (AD) have been identified during recent years in large-scale genome-wide association studies. However, little is known about the mechanisms by which these loci influence AD pathogenesis. OBJECTIVE: To investigate the individual and combined risk effects of the newly identified AD loci. METHODS: Association of 12 AD risk loci with AD and AD-related cerebrospinal fluid (CSF) biomarkers was assessed. Furthermore, a polygenic risk score combining the effect sizes of the top 22 risk loci in AD was calculated for each individual among the clinical and neuropathological cohorts. Effects of individual risk loci and polygenic risk scores were assessed in relation to CSF biomarker levels as well as neurofibrillary pathology and different biochemical measures related to AD pathogenesis obtained from the temporal cortex. RESULTS: Polygenic risk scores associated with CSF amyloid-ß42 (Aß42) levels in the clinical cohort, and with soluble Aß42 levels and γ-secretase activity in the neuropathological cohort. The γ-secretase effect was independent of APOE. APOE-ε4 associated with CSF Aß42 (p < 0.001) levels. For the other risk loci, no significant associations with AD risk or CSF biomarkers were detected after multiple testing correction. CONCLUSIONS: AD risk loci polygenically contribute to Aß pathology in the CSF and temporal cortex, and this effect is potentially associated with increased γ-secretase activity.

Transcriptomics and mechanistic elucidation of Alzheimer's disease risk genes in the brain and in vitro models.

In this study, we have assessed the expression and splicing status of genes involved in the pathogenesis or affecting the risk of Alzheimer's disease (AD) in the postmortem inferior temporal cortex samples obtained from 60 subjects with varying degree of AD-related neurofibrillary pathology. These subjects were grouped based on neurofibrillary pathology into 3 groups: Braak stages 0-II, Braak stages III-IV, and Braak stages V-VI. We also examined the right frontal cortical biopsies obtained during life from 22 patients with idiopathic shunt-responding normal pressure hydrocephalus, a disease that displays similar pathologic alterations as seen in AD. These 22 patients were categorized according to dichotomized amyloid-ß positive or negative pathology in the biopsies. We observed that the expression of FRMD4A significantly decreased, and the expression of MS4A6A significantly increased in relation to increasing AD-related neurofibrillary pathology. Moreover, the expression of 2 exons in both CLU and TREM2 significantly increased with increase in AD-related neurofibrillary pathology. However, a similar trend toward increased expression in CLU and TREM2 was observed with most of the studied exons, suggesting a global change in the expression rather than altered splicing. Correlation of gene expression with well-established AD-related factors, such as α-, ß-, and γ-secretase activities, brain amyloid-ß42 levels, and cerebrospinal fluid biomarkers, revealed a positive correlation between ß-secretase activity and the expression of TREM2 and BIN1. In expression quantitative trait loci analysis, we did not detect significant effects of the risk alleles on gene expression or splicing. Analysis of the normal pressure hydrocephalus biopsies revealed no differences in the expression or splicing profiles of the studied genes between amyloid-ß positive and negative patients. Using the protein-protein interaction-based in vitro pathway analysis tools, we found that downregulation of FRMD4A associated with increased APP-ß-secretase interaction, increased amyloid-ß40 secretion, and altered phosphorylation of tau. Taken together, our results suggest that the expression of FRMD4A, MS4A6A, CLU, and TREM2 is altered in relation to increasing AD-related neurofibrillary pathology, and that FRMD4A may play a role in amyloidogenic and tau-related pathways in AD. Therefore, investigation of gene expression changes in the brain and effects of the identified genes on disease-associated pathways in vitro may provide mechanistic insights on how alterations in these genes may contribute to AD pathogenesis.

High-fat diet increases tau expression in the brain of T2DM and AD mice independently of peripheral metabolic status.

Alzheimer's disease and type 2 diabetes mellitus are risk factors for each other. To investigate the effects of both genetic and high-fat-induced diabetic phenotype on the expression and exon 10 splicing of tau, we used the Alzheimer's disease mouse model (APdE9) cross-bred with the type 2 diabetes mouse model over-expressing insulin-like growth factor 2 in the pancreas. High-fat diet, regardless of the genotype, significantly induced the expression of four repeat tau mRNA and protein in the temporal cortex of female mice. The mRNA levels of three repeat tau were also significantly increased by high-fat diet in the temporal cortex, although three repeat tau expression was considerably lower as compared to four repeat tau. Moreover, high-fat diet significantly increased the mRNA ratio of four repeat tau vs. three repeat tau in the temporal cortex of these mice. All of these effects were independent of the peripheral hyperglycemia, hyperinsulinemia and insulin resistance. Increased four repeat tau and three repeat tau levels significantly associated with impaired memory and reduced rearing in the female mice. High-fat diet did not affect neuroinflammation, Akt/GSK3ß signaling pathway or the expression of tau exon 10 splicing enhancers in the temporal cortex. Our study suggests that the high-fat diet independently of type 2 diabetes or Alzheimer's disease background induces the expression and exon 10 inclusion of tau in the brain of female mice.

Effects of NR1H3 genetic variation on the expression of liver X receptor α and the progression of Alzheimer's disease.

Alzheimer's disease (AD) has been postulated to involve defects in the clearance of amyloid-ß (Aß). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aß. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aß42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aß42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aß42.

Targeting ApoE4/ApoE receptor LRP1 in Alzheimer's disease.

INTRODUCTION: Progressive neuronal loss is a key feature in Alzheimer's disease (AD), which is the most common neurodegenerative disorder in the aging population. Currently, there are no therapeutic means to intervene neuronal damage in AD and therefore innovative approaches to discover novel strategies for the treatment of AD are needed. Based on the prevailing amyloid cascade hypothesis, it is conceivable that lowering the ß-amyloid (Aß) levels is sufficient to slow down the disease process, if started early enough. AREAS COVERED: Here, we review genetic and biological functions related to apolipoprotein E (ApoE) and low-density lipoprotein receptor-related protein 1 receptor (LRP1)-mediated clearance of Aß. Furthermore, we discuss the AD-related therapeutic potential of targeting to ApoE receptor LRP1 at the blood-brain barrier (BBB) and in the periphery. EXPERT OPINION: Due to the recent setbacks in the clinical trials targeting AD, it is instrumental to seek alternative therapeutic approaches, which aim to reduce the accumulation of Aß in the brain tissue. As the ApoE/LRP1-mediated clearance of Aß across the BBB is the key event in the regulation of Aß transcytosis from brain to periphery, direct targeting of this protein entity at the BBB holds a great potential in the treatment of AD.

Negative frequency-dependent selection of sexually antagonistic alleles in Myodes glareolus.

Sexually antagonistic genetic variation, where optimal values of traits are sex-dependent, is known to slow the loss of genetic variance associated with directional selection on fitness-related traits. However, sexual antagonism alone is not sufficient to maintain variation indefinitely. Selection of rare forms within the sexes can help to conserve genotypic diversity. We combined theoretical models and a field experiment with Myodes glareolus to show that negative frequency-dependent selection on male dominance maintains variation in sexually antagonistic alleles. In our experiment, high-dominance male bank voles were found to have low-fecundity sisters, and vice versa. These results show that investigations of sexually antagonistic traits should take into account the effects of social interactions on the interplay between ecology and evolution, and that investigations of genetic variation should not be conducted solely under laboratory conditions.