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Mastroeni, D.

Publications and source records attributed to Mastroeni, D..

11 recordsLinked to original sources

Upregulation of the Proto-Oncogene Src Kinase in Alzheimer's Disease: From Molecular Interactions to Therapeutic Potential

Alzheimers disease (AD) is a progressive neurodegenerative disease, resulting in an irreversible deterioration of multiple brain regions associated with cognitive dysfunction. Phosphorylation of the microtubule-associated protein, Tau, is known to occur decades before symptomatic AD. The Src family of tyrosine kinases are known to phosphorylate select tyrosine sites on Tau and promote microtubule disassembly and subsequent neurofibrillary tangle (NFT) formation. Our data show that the proto-oncogene, non-receptor tyrosine kinase Src colocalizes with a range of late (PHF1) to early (MC1) AD-associated phosphorylated Tau epitopes. The strongest co-occurrence is seen with MC1 (probability of MC1 given Src =100%), an early AD-specific conformational dependent epitope. Single-cell RNA sequencing data of 101 subjects show that Src is upregulated in both AD inhibitory and excitatory neurons. The most significantly affected, by orders of magnitude, were excitatory neurons which are the most prone to pathological Tau accumulation. We measured Src phosphorylation by mass spectrometry across a cohort of 48 patient neocortical tissues and found that Src has increased phosphorylation on Ser75, Tyr187, and Tyr440 in AD, showing that Src kinase undergoes distinct phosphorylation alterations in AD. Through Brownian dynamics simulations of Src and Tau, we show that as Tau undergoes the transition into disease-associated paired helical filaments, there is a notable seven-fold increase in Src contact with Tau. These results collectively emphasize Src kinases central role in Tau phosphorylation and its close association with Tau epitopes, presenting a promising target for potential therapeutic intervention.

neuroscience↗

A public resource of single cell transcriptomes and multiscale networks from persons with and without Alzheimer's disease

The emergence of technologies that can support high-throughput profiling of single cell transcriptomes offers to revolutionize the study of brain tissue from persons with and without Alzheimers disease (AD). Integration of these data with additional complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link observed cell subpopulations and molecular network features within a broader disease-relevant context. We report here single nucleus RNA sequencing (snRNA-seq) profiles generated from superior frontal gyrus cortical tissue samples from 101 exceptionally well characterized, aged subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in peripheral hematological lab parameters, with these observations replicated in an independent, prospective cohort study of ageing and dementia. We also observed an AD-associated CD83(+) microglial subtype with unique molecular networks that encompass many known regulators of AD-relevant microglial biology, and which are associated with immunoglobulin IgG4 production in the transverse colon. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal novel disease biology. The transcriptomic, genetic, phenotypic, and network data resources described within this study are available for access and utilization by the scientific community.

genomics↗

Unveiling a Novel Memory Center in Humans: Neurochemical Identification of the Nucleus Incertus, a Key Pontine Locus Implicated in Stress and Neuropathology

BackgroundThe nucleus incertus (NI) was originally described by Streeter in 1903, as a midline region in the floor of the fourth ventricle (4V) of the human brain with an unknown function. More than a century later, the neuroanatomy of the NI including its forebrain target regions has been described in lower vertebrates, but not in humans. Therefore, we examined the neurochemical anatomy of the human NI using several markers, including the neuropeptide, relaxin-3 (RLN3), and began to explore the distribution of the NI-related RLN3 innervation of the hippocampus. MethodsHistochemical staining of serial, coronal sections (30 {micro}m) of control human postmortem pons was conducted to reveal the presence of the NI by detection of immunoreactivity (IR) for the neuronal marker, microtubule-associated protein-2 (MAP2), two markers present in rat NI, glutamic acid dehydrogenase (GAD)-65/67 and corticotrophin releasing hormone receptor 1 (CRHR1), and RLN3, which is highly expressed in a major population of NI neurons in diverse species. RLN3 and vesicular GABA transporter 1 (vGAT1) mRNA was detected by multiplex, fluorescence in situ hybridization. Postmortem pons sections containing the NI from an Alzheimers disease (AD) case were immunostained for phosphorylated-tau (AT8 antibody), to explore potential relevance to neurodegenerative diseases. Lastly, sections of human hippocampus were stained to detect RLN3-IR and somatostatin (SST)-IR, as SST is expressed in interneurons targeted by RLN3 projections in rodents. ResultsIn the dorsal, anterior-medial region of the human pons, neurons containing RLN3- and MAP2-IR, and RLN3/vGAT1 mRNA-positive neurons were observed in an anatomical pattern consistent with that of the NI in other species. GAD65/67- and CRHR1-immunopositive neurons were also detected within this area. Furthermore, RLN3- and AT8-IR were co-localized within NI neurons of an AD subject. Lastly, RLN3-IR was detected in neurons within the CA1, CA2, CA3, and DG areas of the hippocampus, in the absence of RLN3 mRNA. In the DG, RLN3- and SST-IR were co-localized in a small population of neurons. ConclusionsAspects of the anatomy of the human NI are shared across species, including a population of RLN3-expressing neurons and a RLN3 innervation of the hippocampus. Accumulation of phosphorylated-tau in the NI suggests its possible involvement in AD pathology. Further characterization of the neurochemistry of the human NI will increase our understanding of its functional role in health and disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/556922v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@f4c439org.highwire.dtl.DTLVardef@17e85f5org.highwire.dtl.DTLVardef@18ca7faorg.highwire.dtl.DTLVardef@168246c_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Created with BioRender.com C_FIG

neuroscience↗

Epigenome-wide profiling in the dorsal raphe nucleus highlights cell-type-specific changes in TNXB in Alzheimer's disease

Recent studies have demonstrated that the dorsal raphe nucleus (DRN) is among the first brain regions affected in Alzheimers disease. Hence, in this study we conducted the first comprehensive epigenetic analysis of the DRN in AD, targeting both bulk tissue and single isolated cells. The Illumina Infinium MethylationEPIC BeadChip array was used to analyze the bulk tissue, assessing differentially modified positions (DMoPs) and regions (DMoRs) associated with Braak stage. The strongest Braak stage-associated DMoR in TNXB was targeted in a second patient cohort utilizing single laser-capture microdissected serotonin-positive (5-HT+) and -negative (5-HT-) cells isolated from the DRN. Our study revealed previously identified epigenetic loci, including TNXB and PGLYRP1, and novel loci, including RBMXL2, CAST, GNAT1, MALAT1, and DNAJB13. Strikingly, we found that the methylation profile of TNXB depends both on disease phenotype and cell type analyzed, emphasizing the significance of single cell(-type) neuroepigenetic studies in AD.

neuroscience↗

Modeling of mitochondrial genetic polymorphisms reveals induction of heteroplasmy by pleiotropic disease locus MT:10398A>G

Mitochondrial (MT) dysfunction has been associated with several neurodegenerative diseases including Alzheimers disease (AD). While MT-copy number differences have been implicated in AD, the effect of MT heteroplasmy on AD has not been well characterized. Here, we analyzed over 1,800 whole genome sequencing data from four AD cohorts in seven different tissue types to determine the extent of MT heteroplasmy present. While MT-heteroplasmy was present throughout the entire MT genome for blood samples, we detected MT-heteroplasmy only within the MT control region for brain samples. We observed that an MT variant 10398A>G (rs2853826) was significantly associated with overall MT-heteroplasmy in brain tissue while also being linked with the largest number of distinct disease phenotypes of all annotated MT variants in MitoMap. Using gene-expression data from our brain samples, our modeling discovered several gene networks involved in mitochondrial respiratory chain and Complex I function associated with 10398A>G. The variant was also found to be an expression quantitative trait loci (eQTL) for the gene MT-ND3. We further characterized the effect of 10398A>G by phenotyping a population of lymphoblastoid cell-lines (LCLs) with and without the variant allele. Examination of RNA sequence data from these LCLs reveal that 10398A>G was an eQTL for MT-ND4. We also observed in LCLs that 10398A>G was significantly associated with overall MT-heteroplasmy within the MT control region, confirming the initial findings observed in post-mortem brain tissue. These results provide novel evidence linking MT SNPs with MT heteroplasmy and open novel avenues for the investigation of pathomechanisms that are driven by this pleiotropic disease associated loci.

genomics↗

Chronic Cognitive and Cerebrovascular Function Following Mild Traumatic Brain Injury in Rats

Severe traumatic brain injury results in cognitive dysfunction in part due to vascular perturbations. In contrast, the long-term vasculo-cognitive pathophysiology of mild TBI (mTBI) remains unknown. We evaluated mTBI effects on chronic cognitive and cerebrovascular function and assessed their interrelationships. Sprague-Dawley rats received midline fluid percussion injury (N=20) or sham (N=21). Cognitive function was assessed (3- and 6-month novel object recognition (NOR), novel object location (NOL) and temporal order object recognition (TOR)). 6-month cerebral blood flow (CBF) and blood volume (CBV) using contrast MRI and ex vivo pial artery endothelial and smooth muscle-dependent function were measured. mTBI rats showed impaired NOR, with similar (non-significant) trends in NOL/TOR. Regional CBF and CBV were similar in sham and mTBI. NOR correlated with CBF in lateral hippocampus, medial hippocampus and primary somatosensory barrel cortex while inversely correlating with arterial smooth muscle-dependent dilation. 6-month baseline endothelial and smooth muscle-dependent arterial function were similar among mTBI and sham, but post-angiotensin II stimulation, mTBI showed no change in smooth muscle-dependent dilation from baseline response, unlike the reduction in sham. mTBI led to chronic cognitive dysfunction and altered angiotensin II-stimulated smooth muscle-dependent vasoreactivity, a paradigm that could advance understanding of the long-term sequelae of human mild TBI.

neuroscience↗

Quantitative phosphoproteomics uncovers dysregulated kinase networks in Alzheimer's disease

Alzheimers disease (AD) is a form of dementia characterized by amyloid-{beta} plaques and Tau neurofibrillary tangles that progressively disrupt neural circuits in the brain. The signaling networks underlying the pathological changes in AD are poorly characterized at the level phosphoproteome. Using mass spectrometry, we performed a combined analysis of the tyrosine, serine, and threonine phosphoproteome, and proteome of temporal cortex tissue from AD patients and aged matched controls. We identified several co-correlated peptide modules that were associated with varying levels of phospho-Tau, oligodendrocyte, astrocyte, microglia, and neuronal pathologies in AD patients. We observed phosphorylation sites on kinases targeting Tau as well as other novel signaling factors that were correlated with these peptide modules. Finally, we used a data-driven statistical modeling approach to identify individual peptides and co-correlated signaling networks that were predictive of AD histopathologies. Together, these results build a map of pathology-associated phosphorylation signaling events occurring in AD.

neuroscience↗

Phosphoproteomics identifies microglial Siglec-F inflammatory response during neurodegeneration

Alzheimers disease (AD) is characterized by the appearance of amyloid-{beta} plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK-p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec-F which was upregulated on a subset of reactive microglia. The human paralog Siglec-8 was also upregulated on microglia in AD. Siglec-F and Siglec-8 were upregulated following microglial activation with interferon gamma (IFN{gamma}) in BV-2 cell line and human stem-cell derived microglia models. Siglec-F overexpression activates an endocytic and pyroptotic inflammatory response in BV-2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV-2 cells. Collectively, our results point to an important role for mouse Siglec-F and human Siglec-8 in regulating microglial activation during neurodegeneration. HighlightsO_LIPhosphoproteomics analysis of CK-p25, 5XFAD, and Tau P301S mouse models finds dysregulated signaling networks associated with Alzheimers disease pathologies. C_LIO_LIA phosphorylation site on Siglec-F is found to be upregulated across all three models of disease. C_LIO_LIExpression of Siglec-F and its human paralog Siglec-8 is increased in reactive microglia. C_LIO_LIOverexpression of Siglec-F and Siglec-8 in vitro drives an endocytic and pyroptotic inflammatory response. C_LI In BriefPhosphoproteome signaling changes associated with Alzheimers disease (AD) are poorly characterized. Here, Morshed et al. apply phosphoproteomics to mouse models of AD to uncover a novel microglial receptor, Siglec-F, that is upregulated on a subset of inflammatory microglia across models of neurodegeneration. The human paralog, Siglec-8 is also found to be upregulated in late-onset AD microglia. Overexpression of Siglec-F and related human Siglecs activates pro-inflammatory signaling responses in BV-2 cells.

neuroscience↗

Meta-analysis of epigenome-wide association studies in Alzheime's disease highlights 220 differentially methylated loci across cortex

Epigenome-wide association studies of Alzheimers disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimers disease (N=1,453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N=1,408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further > 600 unique donors. The meta-analysis summary statistics are available in our online data resource (www.epigenomicslab.com/ad-meta-analysis/).

genomics↗

Highly sensitive and multiplexed in situ protein profiling with cleavable fluorescent streptavidin

The ability to perform highly sensitive and multiplexed in situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, here we develop an approach using cleavable biotin conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced by at least 10 fold, compared with the existing methods. After imaging, the fluorophores and the biotins unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be unambiguously detected in individual cell in situ.

systems biology↗

Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

The ability to comprehensively profile proteins in intact tissues in situ is crucial for our understanding of health and disease. However, the existing methods suffer from low sensitivity and limited sample throughput. To address these issues, here we present a highly sensitive and multiplexed in situ protein analysis approach using cleavable fluorescent tyramide and off-the-shelf antibodies. Compared with the current methods, this approach enhances the detection sensitivity and reduces the imaging time by 1-2 orders of magnitude, and can potentially detect hundreds of proteins in intact tissues at the optical resolution. Applying this approach, we studied protein expression heterogeneity in genetically identical cells, and performed expression correlation analysis to identify coregulated proteins. We also profiled >6000 neurons in human formalin-fixed paraffin-embedded (FFPE) hippocampus. By partitioning these neurons into varied cell clusters based on their protein expression profiles, we observed different subregions of the hippocampus consist of neurons from distinct clusters.

systems biology↗