Landscape of brain myeloid cell transcriptome along the spatiotemporal progression of Alzheimer's disease reveals distinct sequential responses to Aß and tau.

Human microglia are critically involved in Alzheimer's disease (AD) progression, as shown by genetic and molecular studies. However, their role in tau pathology progression in human brain has not been well described. Here, we characterized 32 human donors along progression of AD pathology, both in time-from early to late pathology-and in space-from entorhinal cortex (EC), inferior temporal gyrus (ITG), prefrontal cortex (PFC) to visual cortex (V2 and V1)-with biochemistry, immunohistochemistry, and single nuclei-RNA-sequencing, profiling a total of 337,512 brain myeloid cells, including microglia. While the majority of microglia are similar across brain regions, we identified a specific subset unique to EC which may contribute to the early tau pathology present in this region. We calculated conversion of microglia subtypes to diseased states and compared conversion patterns to those from AD animal models. Targeting genes implicated in this conversion, or their upstream/downstream pathways, could halt gene programs initiated by early tau progression. We used expression patterns of early tau progression to identify genes whose expression is reversed along spreading of spatial tau pathology (EC > ITG > PFC > V2 > V1) and identified their potential involvement in microglia subtype conversion to a diseased state. This study provides a data resource that builds on our knowledge of myeloid cell contribution to AD by defining the heterogeneity of microglia and brain macrophages during both temporal and regional pathology aspects of AD progression at an unprecedented resolution.

Distinct transcriptomic responses to Aß plaques, neurofibrillary tangles, and APOE in Alzheimer's disease.

INTRODUCTION: Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE-linked differences remain unclear. METHODS: We performed laser capture microdissection of amyloid beta (Aß) plaques, the 50 µm halo around them, tangles with the 50 µm halo around them, and areas distant (> 50 µm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing. RESULTS: Aß plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aß plaques had more differentially expressed genes than tangles. We identified a gradient Aß plaque > peri-plaque > tangle > distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aß plaques. DISCUSSION: Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aß plaques, and are exacerbated by the APOE ε4 allele.

Endothelial Cells Are Heterogeneous in Different Brain Regions and Are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 human AD and non-AD donors (19 female, 13 male) each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex, and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid ß plaques and cerebral amyloid angiopathy. This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain.SIGNIFICANCE STATEMENT In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

Distinct Transcriptomic Responses to Aß plaques, Neurofibrillary Tangles, and APOE in Alzheimer's Disease.

INTRODUCTION: Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE -linked differences remain unclear. METHODS: We performed laser capture microdissection of Aß plaques, the 50µm halo around them, tangles with the 50µm halo around them, and areas distant (>50µm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing. RESULTS: Aß plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aß plaques had more differentially expressed genes than tangles. We identified a gradient Aß plaque>peri-plaque>tangle>distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aß plaques. DISCUSSION: Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aß plaques, and are exacerbated by the APOE ε4 allele.

Endothelial Cells are Heterogeneous in Different Brain Regions and are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 AD and non-AD donors each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid beta plaques and cerebral amyloid angiopathy (CAA). This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain. Significance Statement: In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

Quantitative comparison of phosphodiesterase mRNA distribution in human brain and peripheral tissues.

Cyclic nucleotide-specific phosphodiesterases (PDEs) play a critical role in signal transduction by regulating the level of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) in cells. The gene expression pattern of a PDE provides important information regarding its role in physiological and pathological processes. In this study, we have established the mRNA expression profile all PDE isoenzymes (PDE1A/B/C, 2A, 3A/B, 4A/B/C/D, 5A, 6A/B/C, 7A/B, 8A/B, 9A, 10A, 11A) in a human cDNA collection consisting of 10 brain regions (parietal, frontal, temporal cortex, hippocampus, striatum, thalamus, hypothalamus, substantia nigra, nucleus accumbens, cerebellum), spinal cord, dorsal root ganglia and 12 peripheral tissues (skeletal muscle, heart, thyroid, adrenal gland, pancreas, bladder, kidney, liver, lung, small intestine, spleen, and stomach). Using quantitative real-time polymerase chain reaction and parallel analysis of a carefully selected group of reference genes, we have determined the relative expression of each PDE isoenzyme across the 24 selected tissues, and also compared the expression of selected PDEs to each other within a given tissue type. Several PDEs show strikingly selective expression (e.g. PDE10A and PDE1B mRNA levels in the caudate nucleus are 20-fold higher than in most other tissues; PDE1C and PDE3A are highly expressed in the heart and PDE8B is expressed very strongly in the thyroid gland). This comprehensive approach provides a coherent and quantitative view of the mRNA expression of the PDE gene family and enables an integration of data obtained with other non-quantitative methods.

Development of homogeneous 384-well high-throughput screening assays for Abeta1-40 and Abeta1-42 using AlphaScreen technology.

Amyloid beta (Abeta) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimer's disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimer's disease research. The current most established assay for analysis of Abeta peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Abeta1-42 and Abeta1-40 using AlphaScreen technology. The assays are capable of detecting Abeta peptides at concentrations <2 pg/mL and, using a final assay volume of 20 microL, routinely generate Z' values >0.85. The AlphaScreen format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen technology permits robust, efficient, and cost-effective quantitation of Abeta peptides.

The antianginal agent trimetazidine does not exert its functional benefit via inhibition of mitochondrial long-chain 3-ketoacyl coenzyme A thiolase.

Trimetazidine acts as an effective antianginal clinical agent by modulating cardiac energy metabolism. Recent published data support the hypothesis that trimetazidine selectively inhibits long-chain 3-ketoacyl CoA thiolase (LC 3-KAT), thereby reducing fatty acid oxidation resulting in clinical benefit. The aim of this study was to assess whether trimetazidine and ranolazine, which may also act as a metabolic modulator, are specific inhibitors of LC 3-KAT. We have demonstrated that trimetazidine and ranolazine do not inhibit crude and purified rat heart or recombinant human LC 3-KAT by methods that both assess the ability of LC 3-KAT to turnover specific substrate, and LC 3-KAT activity as a functional component of intact cellular beta-oxidation. Furthermore, we have demonstrated that trimetazidine does not inhibit any component of beta-oxidation in an isolated human cardiomyocyte cell line. Ranolazine, however, did demonstrate a partial inhibition of beta-oxidation in a dose-dependent manner (12% at 100 micromol/L and 30% at 300 micromol/L). Both trimetazidine (10 micromol/L) and ranolazine (20 micromol/L) improved the recovery of cardiac function after a period of no flow ischemia in the isolated working rat heart perfused with a buffer containing a relatively high concentration (1.2 mmol/L) of free fatty acid. In summary, both trimetazidine and ranolazine were able to improve ischemic cardiac function but inhibition of LC 3-KAT is not part of their mechanism of action. The full text of this article is available online at http://www.circresaha.org.

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface.

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as "shedding". The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20-40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4 degrees C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

Mitogen-activated protein kinase kinase 7 is activated during low potassium-induced apoptosis in rat cerebellar granule neurons.

A stress-activated protein kinase pathway comprising mitogen-activated protein kinase kinases (MKKs), c-Jun N-terminal kinase (JNK) and the transcription factor c-Jun is implicated in neuronal apoptosis. Using an immune-complex kinase assay, we measured the activation of MKK4 and MKK7 in low potassium (LK)-induced apoptosis of rat cerebellar granule neurons (CGN). MKK7, but not MKK4, was activated within the first 4-6 h in four independent sets of 14-h CGN apoptosis time-courses. CEP-1347 (500 nM), an mixed-lineage kinase 3 inhibitor, prevented MKK7 activation and cell death following exposure of CGN cultures to LK-induced apoptosis. Western blot analysis revealed that levels of phosphorylated c-Jun were elevated between 30 min and 10 h of CGN apoptosis, temporally consistent with MKK7 activation. These data suggest that MKK7 is responsible for activating the JNK pathway during LK-induced CGN apoptosis.