Alzheimer's Disease Amyloid Plaques Research Articles

Association between hypothalamic Alzheimer's disease pathology and body mass index: The Hisayama study.

The hypothalamus is the region of the brain that integrates the neuroendocrine system and whole-body metabolism. Patients with Alzheimer's disease (AD) have been reported to exhibit pathological changes in the hypothalamus, such as neurofibrillary tangles (NFTs) and amyloid plaques (APs). However, few studies have investigated whether hypothalamic AD pathology is associated with clinical factors. We investigated the association between AD-related pathological changes in the hypothalamus and clinical pictures using autopsied brain samples obtained from deceased residents of a Japanese community. A total of 85 autopsied brain samples were semi-quantitatively analyzed for AD pathology, including NFTs and APs. Our histopathological studies showed that several hypothalamic nuclei, such as the tuberomammillary nucleus (TBM) and lateral hypothalamic area (LHA), are vulnerable to AD pathologies. NFTs are observed in various neuropathological states, including normal cognitive cases, whereas APs are predominantly observed in AD. Regarding the association between hypothalamic AD pathologies and clinical factors, the degree of APs in the TBM and LHA was associated with a lower body mass index while alive, after adjusting for sex and age at death. However, we found no significant association between hypothalamic AD pathology and the prevalence of hypertension, diabetes, or dyslipidemia. Our study showed that a lower BMI, which is a poor prognostic factor of AD, might be associated with hypothalamic AP pathology and highlighted new insights regarding the disruption of the brain-whole body axis in AD.

Accumulation of neutral lipids in dystrophic neurites surrounding amyloid plaques in Alzheimer's disease

Alzheimer's disease (AD) is characterized by the formation β-amyloid (Aβ) deposited neuritic plaques. Recent evidence suggests that abnormal lipid metabolism and accumulation could serve as biomarkers for neurodegenerative diseases, including AD. Tubular endoplasmic reticulum protein, reticulon 3 (RTN3), plays a crucial role in the development of neuritic plaque and lipid metabolism in AD brains. In present study, we sought to investigate a potential association between neutral lipid accumulation and AD pathology. BODIPY 500/510 dye was used to label neutral lipid surrounding Aβ plaques in APPNL-G-F mouse and AD postmortem brains samples. Immunofluorescent images were captured using confocal microscope and co-localization between lipid metabolism proteins and neutral lipids were evaluated. Lipid accumulation in Aβ plaque surrounding dystrophic neurites (DNs) was observed in the cortical region of AD mouse models and human AD brain samples. The neutral lipid staining was not co-localized with IBA1-labeled microglia or GFAP-labeled astrocytes, but it was co-labeled with VAMP2 and neurofilament. We further showed that neutral lipids were accumulated in RTN3 immunoreactive DNs. Both the neutral lipids accumulation and RIDNs formation showed age-dependent patterns in surrounding amyloid plaques. Mechanistic studies revealed that RTN3 likely contributes to the enrichment of neutral lipids near plaques by interacting with heat shock cognate protein 70 (HSC70) and diminishing its function in chaperone-mediated lipophagy. Our study provides immunohistochemical evidence of neutral lipids being enriched in DNs near amyloid plaques. Our findings shed light on RTN3-mediaed lipid accumulation in AD neuropathology and provide fresh insights into the role of RTN3 in neurodegenerative diseases.

Flavonoids and fibrate modulate apoE4-induced processing of amyloid precursor protein in neuroblastoma cells.

Apolipoprotein (apo) E4, being a major genetic risk factor for Alzheimer's disease (AD), is actively involved in the proteolytic processing of amyloid precursor protein (APP) to amyloid β (Aβ) peptide, the principle constituent of amyloid plaques in Alzheimer Disease (AD) patients. ApoE4 is believed to affect APP processing through intracellular cholesterol homeostasis, whereas lowering the cholesterol level by pharmacological agents has been suggested to reduce Aβ production. This study has investigated the effects of hypolipidemic agents fenofibrate, and the flavonoids-naringenin and diosmetin-on apoE4-induced APP processing in rat neuroblastoma cells stably transfected with human wild-type APP 695 (B103-hAPP695wt). B103-hAPP695wt cells were pretreated with different doses of flavonoids and fenofibrate for 1 h prior to apoE4 exposure for 24 h. ApoE4-induced production of intra- and extracellular Aβ peptides has been reduced with fenofibrate, naringenin, and diosmetin treatments. Pretreatment with diosmetin has significantly reduced apoE4-induced full-length APP (fl- APP) expression, whereas naringenin and fenofibrate had no effect on it. In addition, the increase in the apoE4-induced secretion of sAPPtotal and sAPPα has been dose-dependently reduced with drug pretreatment. On the other hand, the decrease in the expression of both APP-carboxy terminal fragments (CTF)-α and -β (generated by the α- or β-secretase cleavage of APP) by apoE4 was dose-dependently increased in cells pretreated with fenofibrate and naringenin but not diosmetin. Thus, we suggest that fenofibrate, naringenin, and diosmetin treatments can reduce apoE4- induced Aβ production by distinct mechanisms that may prove useful in developing drugs for AD patients.

Probing Chemical Complexity of Amyloid Plaques in Alzheimer's Disease Mice using Hyperspectral Raman Imaging.

One of the distinctive pathological features of Alzheimer's disease (AD) is the deposition of amyloid plaques within the brain of affected individuals. These plaques have traditionally been investigated using labeling techniques such as immunohistochemical imaging. However, the use of labeling can disrupt the structural integrity of the molecules being analyzed. Hence, it is imperative to employ label-free imaging methods for noninvasive examination of amyloid deposits in their native form, thereby providing more relevant information pertaining to AD. This study presents compelling evidence that label-free and nondestructive confocal Raman imaging is a highly effective approach for the identification and chemical characterization of amyloid plaques within cortical regions of an arcAβ mouse model of AD. Furthermore, this investigation elucidates how the spatial correlation of Raman signals can be exploited to identify robust Raman marker bands and discern proteins and lipids from amyloid plaques. Finally, this study uncovers the existence of distinct types of amyloid plaques in the arcAβ mouse brain, exhibiting significant disparities in terms of not only shape and size but also molecular composition.

Transferrin-Conjugated Melittin-Loaded L-Arginine-Coated Iron Oxide Nanoparticles for Mitigating Beta-Amyloid Pathology of the 5XFAD Mouse Brain.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases and a major contributor to dementia. Although the cause of this condition has been identified long ago as aberrant aggregations of amyloid and tau proteins, effective therapies for it remain elusive. The complexities of drug development for AD treatment are often compounded by the impermeable blood-brain barrier and low-yield brain delivery. In addition, the use of high drug concentrations to overcome this challenge may entail side effects. To address these challenges and enhance the precision of delivery into brain regions affected by amyloid aggregation, we proposed a transferrin-conjugated nanoparticle-based drug delivery system. The transferrin-conjugated melittin-loaded L-arginine-coated iron oxide nanoparticles (Tf-MeLioNs) developed in this study successfully mitigated melittin-induced cytotoxicity and hemolysis in the cell culture system. In the 5XFAD mouse brain, Tf-MeLioNs remarkably reduced amyloid plaque accumulation, particularly in the hippocampus. This study suggested Tf-LioNs as a potential drug delivery platform and Tf-MeLioNs as a candidate for therapeutic drug targeting of amyloid plaques in AD. These findings provide a foundation for further exploration and advancement in AD therapeutics.

Proteomics of the astrocyte secretome reveals changes in their response to soluble oligomeric Aβ.

Astrocytes associate with amyloid plaques in Alzheimer's disease (AD). Astrocytes react to changes in the brain environment, including increasing concentrations of amyloid-β (Aβ). However, the precise response of astrocytes to soluble small Aβ oligomers at concentrations similar to those present in the human brain has not been addressed. In this study, we exposed astrocytes to media from neurons that express the human amyloid precursor protein (APP) transgene with the double Swedish mutation (APPSwe), and which contains APP-derived fragments, including soluble human Aβ oligomers. We then used proteomics to investigate changes in the astrocyte secretome. Our data show dysregulated secretion of astrocytic proteins involved in the extracellular matrix and cytoskeletal organization and increase secretion of proteins involved in oxidative stress responses and those with chaperone activity. Several of these proteins have been identified in previous transcriptomic and proteomic studies using brain tissue from human AD and cerebrospinal fluid (CSF). Our work highlights the relevance of studying astrocyte secretion to understand the brain response to AD pathology and the potential use of these proteins as biomarkers for the disease.

RO6807936 as a Novel PET Radiotracer for in Vitro and in Vivo Visualization and Quantification of BACE1 in the Rodent and Baboon Brain.

The beta-site amyloid precursor protein cleaving enzyme (BACE1) is responsible for initiating the generation of beta-amyloid, the major constituent of amyloid plaques in Alzheimer's disease (AD). The purpose of this study was to develop a specific BACE1 radioligand for visualization of the distribution pattern and quantification of the BACE1 protein in the rodent and monkey brain both in vitro by autoradiography and in vivo by positron emission tomography (PET). The BACE1 inhibitor RO6807936 originating from an in-house chemical drug optimization program was selected based on its PET tracer like physicochemical properties and a favorable pharmacokinetic profile. Saturation binding analysis of [3 H]RO6807936 revealed specific and high affinity binding (KD = 2.9 nM) and a low Bmax value (4.3 nM) of the BACE1 protein in native rat brain membranes. [3 H]RO6807936 binding showed a ubiquitous distribution on rat brain slices in vitro with higher levels in the CA3 8 cell layer and the granule cell layer of the hippocampus. In a next step, RO6807936 was successfully radiolabelled with carbon-11 and showed acceptable uptake in the baboon brain as well as a widespread and rather homogeneous distribution consistent with rodent data. In vivo blockade studies with a specific BACE1 inhibitor reduced uptake of the tracer to homogenous levels across brain regions and demonstrated specificity of the signal. Our data warrant further profiling of this PET tracer candidate in humans to investigate BACE1 expression in normal individuals and those with AD and as an imaging biomarker for target occupancy studies in clinical drug trials.

Human platelets release amyloid peptides β1-40 and β1-42 in response to hemostatic, immune, and hypoxic stimuli

BackgroundPlatelets contain high levels of amyloid β (Aβ) peptides and have been suggested to participate in the deposition of amyloid plaques in Alzheimer’s disease. ObjectivesThis study aimed to determine whether human platelets release pathogenic Aβ peptides Aβ1-42 and Aβ1-40 and to characterize the mechanisms regulating this phenomenon. MethodsEnzyme-linked immunosorbent assays (ELISAs) and single platelet immunostaining were utilised to study the release and subcellular localisation of Aβ, respectively. ResultsELISAs revealed that thrombin and the pro-inflammatory molecule lipopolysaccharide (LPS) induce platelet release of both Aβ1-42 and Aβ1-40. Notably, LPS preferentially induced the release of Aβ1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective β secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aβ1-40 or Aβ1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aβ peptides with platelet alpha granules. ConclusionTaken together, our data suggest that human platelets release pathogenic Aβ peptides as a result of a store-and-release mechanism rather than a de novo proteolytic event. Although further studies are required to fully characterize this phenomenon, we suggest the possibility of a role for platelets in the deposition of Aβ peptides and the formation of amyloid plaques. Interestingly, the combination of hypoxia and inflammation that we simulated in vitro with reduced oxygen tension and LPS may increase the release of fibrillogenic Aβ1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of patients with Alzheimer’s disease.

Monocyte-derived cells invade brain parenchyma and amyloid plaques in human Alzheimer’s disease hippocampus

Microglia are brain-resident myeloid cells and play a major role in the innate immune responses of the CNS and the pathogenesis of Alzheimer's disease (AD). However, the contribution of nonparenchymal or brain-infiltrated myeloid cells to disease progression remains to be demonstrated. Here, we show that monocyte-derived cells (MDC) invade brain parenchyma in advanced stages of AD continuum using transcriptional analysis and immunohistochemical characterization in post-mortem human hippocampus. Our findings demonstrated that a high proportion (60%) of demented Braak V–VI individuals was associated with up-regulation of genes rarely expressed by microglial cells and abundant in monocytes, among which stands the membrane-bound scavenger receptor for haptoglobin/hemoglobin complexes or Cd163. These Cd163-positive MDC invaded the hippocampal parenchyma, acquired a microglial-like morphology, and were located in close proximity to blood vessels. Moreover, and most interesting, these invading monocytes infiltrated the nearby amyloid plaques contributing to plaque-associated myeloid cell heterogeneity. However, in aged-matched control individuals with hippocampal amyloid pathology, no signs of MDC brain infiltration or plaque invasion were found. The previously reported microglial degeneration/dysfunction in AD hippocampus could be a key pathological factor inducing MDC recruitment. Our data suggest a clear association between MDC infiltration and endothelial activation which in turn may contribute to damage of the blood brain barrier integrity. The recruitment of monocytes could be a consequence rather than the cause of the severity of the disease. Whether monocyte infiltration is beneficial or detrimental to AD pathology remains to be fully elucidated. These findings open the opportunity to design targeted therapies, not only for microglia but also for the peripheral immune cell population to modulate amyloid pathology and provide a better understanding of the immunological mechanisms underlying the progression of AD.

A protein aggregation platform that distinguishes oligomers from amyloid fibrils.

Deposition of aggregated proteins is a pathological feature in many neurodegenerative disorders such as Alzheimer's and Parkinson's. In addition to insoluble amyloid fibrils, protein aggregation leads to the formation of soluble oligomers, which are more toxic and pathogenic than fibrils. However, it is challenging to screen for inhibitors targeting oligomers due to the overlapping processes of oligomerization and fibrillization. Here we report a protein aggregation platform that uses intact and split TEM-1 β-lactamase proteins as reporters of protein aggregation. The intact β-lactamase fused with an amyloid protein can report the overall protein aggregation, which leads to loss of lactamase activity. On the other hand, reconstitution of active β-lactamase from the split lactamase construct requires the formation of amyloid oligomers, making the split lactamase system sensitive to oligomerization. Using Aβ, a protein that forms amyloid plaques in Alzheimer's disease, we show that the growth curves of bacterial cells expressing either intact or split lactamase-Aβ fusion proteins can report changes in the Aβ aggregation. The cell lysate lactamase activity assays show that the oligomer fraction accounts for 20% of total activity for the split lactamase-Aβ construct, but only 3% of total activity for the intact lactamase-Aβ construct, confirming the sensitivity of the split lactamase to oligomerization. The combination of the intact and split lactamase constructs allows the distinction of aggregation modulators targeting oligomerization from those targeting overall aggregation. These low-cost bacterial cell-based and biochemical assays are suitable for high-throughput screening of aggregation inhibitors targeting oligomers of various amyloid proteins.

Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss.

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.

Single cell biology to analyse cellular heterogeneity in the response to amyloid plaques in Alzheimer’s Disease

AbstractMicroglial activation and neuroinflammation are crucial in the pathogenesis of Alzheimer’s disease. However, studies in mouse models and human postmortem samples have yielded divergent results regarding transcriptional programs in microglia elicited by exposure to amyloid‐b pathology. Here, we investigate 127,000 single cell expression profiles of human microglia isolated freshly from a xenotransplantation model for AD. Human microglia adopt a different, more complex spectrum of cell states than mouse microglia. Besides a disease associated (DAM) profile, human microglia show a pronounced HLA‐response related to antigen presentation in response to amyloid‐b plaques, which is absent in mouse. Furthermore, oligomeric amyloid‐β present in this model induces a distinctive pro‐inflammatory CRM response characterized by the expression of cytokines and chemokines, also not seen in mice. TREM2 and APOE polymorphisms modulate the response of microglia to amyloid‐b plaques, much less to oligomeric Aβ. In conclusion, human microglia mount a multipronged ARM response to amyloid pathology in Alzheimer’s disease. This implies that therapeutic strategies targeting microglia need to analyse carefully the effect on these different cell states some of which seem protective.

APOE genotype and biological sex regulate astroglial interactions with amyloid plaques in Alzheimer’s disease mice

The most significant genetic risk factor for developing late-onset Alzheimer’s disease (AD) is the ε4 allele of apolipoprotein E (APOE4). APOE genotype and biological sex are key modulators of microglial and astroglial function, which exert multiple effects on AD pathogenesis. Here, we show astroglial interactions with amyloid plaques in the EFAD transgenic mouse model of AD. Using confocal microscopy, we observed significantly lower levels of astrocytic plaque coverage and plaque compaction (beneficial effects of glial barrier formation) with APOE4 genotype and female sex. Conversely, neurite damage and astrocyte activation in the plaque environment were significantly higher in APOE4 carriers and female mice. Astrocyte coverage of plaques was highest in APOE3 males and poorest in APOE4 females. Collectively, our findings provide new insights into the roles of astroglia and highlight the importance of addressing independent and interactive effects of APOE genotype and biological sex in understanding processes contributing to AD pathogenesis.

Translational regulation by ribosome-associated quality control in neurodegenerative disease, cancer, and viral infection.

Translational control at the initiation, elongation, and termination steps exerts immediate effects on the rate as well as the spatiotemporal dynamics of new protein synthesis, shaping the composition of the proteome. Translational control is particularly important for cells under stress as during viral infection or in disease conditions such as cancer and neurodegenerative diseases. Much has been learned about the control mechanisms acting at the translational initiation step under normal or pathological conditions. However, problems during the elongation or termination steps of translation can lead to ribosome stalling and ribosome collision, which will trigger ribosome-associated quality control (RQC) mechanism. Inadequate RQC may lead to the accumulation of faulty translation products that perturb protein homeostasis (proteostasis). Proteostasis signifies a cellular state in which the synthesis, folding, and degradation of proteins are maintained at a homeostatic state such that an intact proteome is preserved. Cellular capacity to preserve proteostasis declines with age, which is thought to contribute to age-related diseases. Proteostasis failure manifested as formation of aberrant protein aggregates, epitomized by the amyloid plaques in Alzheimer’s disease (AD), is a defining feature of neurodegenerative diseases. The root cause of the proteostasis failure and protein aggregation is still enigmatic. Here I will review recent studies supporting that faulty translation products resulting from inadequate RQC of translational stalling and ribosome collision during the translation of problematic mRNAs can be the root cause of proteostasis failure and may represent novel therapeutic targets for neurodegenerative diseases. I will also review evidence that translation regulation by RQC is operative in cancer cells and during viral infection. Better understanding of RQC mechanism may lead to novel therapeutic strategies against neurodegenerative diseases, cancer, and viral infections, including the ongoing COVID-19 pandemic.

Nanoscale Infrared Spectroscopy Identifies Structural Heterogeneity in Individual Amyloid Fibrils and Prefibrillar Aggregates.

Amyloid plaques are one of the central manifestations of Alzheimer's disease pathology. Aggregation of the amyloid beta (Aβ) protein from amorphous oligomeric species to mature fibrils has been extensively studied. However, structural heterogeneities in prefibrillar species, and how that affects the structure of later-stage aggregates are not yet well understood. The integration of infrared spectroscopy with atomic force microscopy (AFM-IR) allows for identifying the signatures of individual nanoscale aggregates by spatially resolving spectra. We use AFM-IR to demonstrate that amyloid oligomers exhibit significant structural variations as evidenced in their infrared spectra. This heterogeneity is transmitted to and retained in protofibrils and fibrils. We show that amyloid fibrils do not always conform to their putative ordered structure and structurally different domains exist in the same fibril. We further demonstrate that these structural heterogeneities manifest themselves as a lack of β sheet structure in amyloid plaques in Alzheimer's tissue using infrared imaging.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid β Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid β (Aβ) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non‐protein components of Aβ deposits that not only modulates Aβ aggregation, but also acts as a receptor for Aβ fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aβ could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6‐O‐phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP‐mediated Aβ fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Label-free Raman and fluorescence imaging of amyloid plaques in human Alzheimer’s disease brain tissue reveal carotenoid accumulations

Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by the presence of extracellular deposits (plaques) of amyloid-beta peptide and intracellular aggregates of phosphorylated tau. In general, these hallmarks are studied by techniques requiring chemical pre-treatment and indirect labeling. Imaging techniques that require no labeling and could be performed on tissue in its native form could contribute to a better understanding of the disease. In this article a combination of label-free and non-invasive techniques is presented to study the biomolecular composition of AD human brain tissue. We build on previous research that already revealed the autofluorescence property of plaque, and the presence of carotenoids in cored plaques. Here, we present further results on cored plaques: showing blue and green autofluorescence emission coming from the same plaque location. Raman microscopy was used to confirm the presence of carotenoids in the plaque areas, with clear peaks around 1150 and 1514 cm−1. Carotenoid reference spectra were recorded in hexane solution, but also adsorbed on aggregated Aβ42 peptides; the latter agreed better with the Raman spectra observed in plaques. From the six single carotenoids measured, lycopene matched closest with the peak positions observed in the cored plaques. Lastly, stimulated Raman scattering (SRS) microscopy measurements were performed, targeting the shift of the beta-sheet Amide I peak observed in plaques. Employing SRS in the C–H stretch region we also looked for the presence of a lipid halo around plaque, as reported in the literature for transgenic AD mice, but such a halo was not observed in these human AD brain samples.

PLCG2 is associated with the inflammatory response and is induced by amyloid plaques in Alzheimer\u2019s disease

BackgroundAlzheimer’s disease (AD) is characterized by robust microgliosis and phenotypic changes that accompany disease pathogenesis. Accumulating evidence from genetic studies suggests the importance of phospholipase C γ 2 (PLCG2) in late-onset AD (LOAD) pathophysiology. However, the role of PLCG2 in AD is still poorly understood.MethodsUsing bulk RNA-Seq (N=1249) data from the Accelerating Medicines Partnership-Alzheimer’s Disease Consortium (AMP-AD), we investigated whether PLCG2 expression increased in the brains of LOAD patients. We also evaluated the relationship between PLCG2 expression levels, amyloid plaque density, and expression levels of microglia specific markers (AIF1 and TMEM119). Finally, we investigated the longitudinal changes of PLCG2 expression in the 5xFAD mouse model of AD. To further understand the role of PLCG2 in different signaling pathways, differential gene expression and co-expression network analyses were performed using bulk RNA-Seq and microglial single-cell RNA-Seq data. To substantiate the human analyses, we performed differential gene expression analysis on wild-type (WT) and inactivated Plcg2 mice and used immunostaining to determine if the differentially expressed genes/pathways were altered by microglial cell coverage or morphology.ResultsWe observed significant upregulation of PLCG2 expression in three brain regions of LOAD patients and significant positive correlation of PLCG2 expression with amyloid plaque density. These findings in the human brain were validated in the 5xFAD amyloid mouse model, which showed disease progression-dependent increases in Plcg2 expression associated with amyloid pathology. Of note, increased Plcg2 expression levels in 5xFAD mice were abolished by reducing microglia. Furthermore, using bulk RNA-Seq data, we performed differential expression analysis by comparing cognitively normal older adults (CN) with 75th percentile (high) and 25th percentile (low) PLCG2 gene expression levels to identify pathways related to inflammation and the inflammatory response. The findings in the human brain were validated by differential expression analyses between WT and plcg2 inactivated mice. PLCG2 co-expression network analysis of microglial single-cell RNA-Seq data identified pathways related to the inflammatory response including regulation of I-kappaB/NF-kappa B signaling and response to lipopolysaccharide.ConclusionsOur results provide further evidence that PLCG2 plays an important role in AD pathophysiology and may be a potential target for microglia-targeted AD therapies.

Does Soluble TREM2 Protect Against Alzheimer's Disease?

Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) is a pattern recognition receptor on myeloid cells, and is upregulated on microglia surrounding amyloid plaques in Alzheimer's disease (AD). Rare, heterozygous mutations in TREM2 (e.g., R47H) increase AD risk several fold. TREM2 can be cleaved at the plasma membrane by metalloproteases to release the ectodomain as soluble TREM2 (sTREM2). Wild-type sTREM2 binds oligomeric amyloid beta (Aβ) and acts as an extracellular chaperone, blocking and reversing Aβ oligomerization and fibrillization, and preventing Aβ-induced neuronal loss in vitro. Whereas, R47H sTREM2 increases Aβ fibrillization and neurotoxicity. AD brains expressing R47H TREM2 have more fibrous plaques with more neuritic pathology around these plaques, consistent with R47H sTREM2 promoting Aβ fibrillization relative to WT sTREM2. Brain expression or injection of wild-type sTREM2 reduces pathology in amyloid models of AD in mice, indicating that wild-type sTREM2 is protective against amyloid pathology. Levels of sTREM2 in cerebrospinal fluid (CSF) fall prior to AD, rise in early AD, and fall again in late AD. People with higher sTREM2 levels in CSF progress more slowly into and through AD than do people with lower sTREM2 levels, suggesting that sTREM2 protects against AD. However, some of these experiments can be interpreted as full-length TREM2 protecting rather than sTREM2, and to distinguish between these two possibilities, we need more experiments testing whether sTREM2 itself protects in AD and AD models, and at what stage of disease. If sTREM2 is protective, then treatments could be designed to elevate sTREM2 in AD.

Modulating physicochemical properties of tetrahydropyridine-2-amine BACE1 inhibitors with electron-withdrawing groups: A systematic study

A common challenge for medicinal chemists is to reduce the pKa of strongly basic groups' conjugate acids into a range that preserves the desired effects, usually potency and/or solubility, but avoids undesired effects like high volume of distribution (Vd), limited membrane permeation, and off-target binding to, notably, the hERG channel and monoamine receptors. We faced this challenge with a 3,4,5,6-tetrahydropyridine-2-amine scaffold harboring an amidine, a key structural component of potential inhibitors of BACE1, the rate-limiting enzyme in the production of Aβ species that make up amyloid plaques in Alzheimer's disease. In our endeavor to balance potency with desirable properties to achieve brain penetration, we introduced a diverse set of groups in beta position of the amidine that modulate logD, PSA and pKa. Given the synthetic challenge to prepare these highly functionalized warheads, we first developed a design flow including predicted physicochemical parameters which allowed us to select only the most promising candidates for synthesis. For this we evaluated a set of commercial packages to predict physicochemical properties, which can guide medicinal chemists in their endeavors to modulate pKa values of amidine and amine bases.