Biology subjects

Denham, M.

Publications and source records attributed to Denham, M..

3 recordsLinked to original sources

bioRxiv2024

MJF-14 proximity ligation assay detects early non-inclusion alpha-synuclein pathology with enhanced specificity and sensitivity

Lewy pathology, consisting of Lewy bodies and Lewy neurites, is the pathological hallmark of synucle-inopathies such as Parkinsons disease and dementia with Lewy bodies, but it is generally thought to represent late-stage pathological changes. In contrast, -synuclein oligomers are regarded as early-stage pathology, likely involved in disease progression and cellular toxicity. Oligomers, however, are not de-tected by standard immunohistochemistry but require specific detection techniques such as the proxim-ity ligation assay (PLA). Here, we describe the MJF-14 PLA, a new PLA towards aggregated -synuclein with unprecedented specificity, attained by the utilization of aggregate conformation-specific -synu-clein antibody MJFR-14-6-4-2 (hereafter MJF-14). Signal in the assay directly correlates with -synuclein aggregation in SH-SY5Y cells, as treatment with aggregation inhibitor ASI1D significantly lowers PLA sig-nal. In human cortical neurons, MJF-14 PLA detects pre-formed fibril-induced aggregation, especially prominent when using stealth PFFs invisible to the MJF-14 antibody. Co-labelling of MJF-14 PLA and pS129--synuclein immunofluorescence in post-mortem dementia with Lewy bodies cases showed that while the MJF-14 PLA reveals extensive non-inclusion pathology, it is not sensitive towards Lewy bodies. In Parkinsons disease brain, direct comparison of PLA and IHC with the MJF-14 antibody, combined with machine learning-based quantification, showed striking -synuclein pathology preceding the formation of conventional Lewy pathology. The majority of the PLA-revealed non-inclusion pathology was found in the neuropil, including some clearly located in the presynaptic terminals. With this work, we introduce an improved -synuclein aggregate PLA to uncover abundant non-inclusion pathology, which deserves future validation with multiple brain bank resources and in different synucleinopathies.

neuroscience↗

bioRxiv2024

APP and its intracellular domain modulate Alzheimers disease risk gene networks in transgenic APPsw and PSEN1M146I porcine models

Alzheimers disease (AD) is a progressive neurodegenerative disorder and the most frequent cause of dementia. The disease has a substantial genetic component comprising both highly penetrant familial mutations (APP, PSEN1, and PSEN2) and sporadic cases with complex genetic etiology. Mutations in APP and PSEN1/2 alter the proteolytic processing of APP to its metabolites, including A{beta} and APP Intracellular Domain (AICD). In this study, we use transgenic porcine models carrying the human APPsw and PSEN1M146I transgenes to demonstrate the pathobiological relevance of transcriptional regulation facilitated by APP and its AICD domain. Through molecular characterization of hippocampal tissue, we describe the differential expression of gene sets that cluster in molecular pathways with translational relevance to AD. We further identify phosphorylated and unphosphorylated AICD in differential complexes with proteins implicated in signal transduction and transcriptional regulation. Integrative genomic analysis of transcriptional changes in somatic cell cultures derived from pigs treated with {gamma}-secretase inhibitor demonstrates the importance of {gamma}-secretase APP processing in transcriptional regulation. Our data supports a model in which APP and, in particular, its AICD domain, modulates gene networks associated with AD pathobiology through interaction with signaling proteins. One Sentence SummaryUtilizing transgenic porcine models, our study reveals that Alzheimers disease-related mutations affect neuronal gene expression and highlights the role of the AICD domain of APP in modulating gene networks associated with Alzheimers pathobiology.

cell biology↗

bioRxiv2021

Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

The differentiation of human pluripotent stem cells (hPSCs) into mesencephalic dopaminergic (mesDA) neurons requires a precise combination of extrinsic factors that recapitulates the in vivo environment and timing. Current methods are capable of generating authentic mesDA neurons after long-term culture in vitro; however, when mesDA progenitors are transplanted in vivo, the resulting mesDA neurons are only minor components of the graft. This low yield hampers the broad use of these cells in the clinic. In this study, we genetically modified pluripotent stem cells to generate a novel type of stem cells called lineage-restricted undifferentiated stem cells (LR-USCs), which robustly generate mesDA neurons. LR-USCs are prevented from differentiating into a broad range of nondopaminergic cell types by knocking out genes that are critical for the specification of cells of alternate lineages. Specifically, we target transcription factors involved in the production of spinal cord and posterior hindbrain cell types. When LR-USCs are differentiated under caudalizing condition, which normally give rise to hindbrain cell types, a large proportion adopt a midbrain identity and develop into authentic mesDA neurons. We show that the mesDA neurons are electrophysiologically active, and due to their higher purity, are capable of restoring motor behavior eight weeks after transplantation into 6-hydroxydopamine (6-OHDA)-lesioned rats. This novel strategy improves the reliability and scalability of mesDA neuron generation for clinical use.

neuroscience↗