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  into a healthy control hiPSC line via CRISPR-Cas9 precision gene editing. These hiPSC were differentiated into microglia, to evaluate the pro-inflammatory profile and metabolic state. Moreover, hiPSC- derived neurons have been cultured with conditioned microglia media to investigate disease specific interactions between the two cell populations. Interestingly, we identified two divergent inflammatory microglial phenotypes resulting from the underlying mutations, indicated by a severe pro-inflammatory profile in CHMP2B homozygous FTD3 microglia, and an “unresponsive” CHMP2B heterozygous FTD3 microglial state. These findings correlate with our observations of increased phagocytotic activity in CHMP2B homozygous FTD3, and impaired protein degradation in CHMP2B heterozygous microglia. Metabolic mapping revealed a metabolic reprogramming of the CHMP2B heterozygous FTD3 microglia, favoring glycolysis, and a compensatory up-regulation in glutamine metabolism in the CHMP2B homozygous microglia. Intriguingly, conditioned CHMP2B homozygous FTD3 microglia media displayed neurotoxic effects, which was not evident for the heterozygous microglia. Strikingly, IFN-γ treatment initiated an immune boost of the CHMP2B heterozygous FTD3 microglia, that promoted neural growth. Our findings indicate that the microglial profile, activity and behavior is highly dependent on the status of the CHMP2B mutation. We propose that the heterozygous state of the mutation in FTD3 patients could potentially be exploited in form of immune-boosting intervention strategies to counteract neurodegeneration.
Conclusions: Our results suggest that oIAPP induces contraction of brain pericytes and that pramlintide can reverse such contraction. oIAPP may compromise cerebral blood flow and reduce oxygen supply, eventually causing pericyte death and impairing the function of the blood-brain barrier.
Glial and vascular contributions to neurodegenerative diseases
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Long-read RNA sequencing reveals new and diverse genes and RNA isoforms expressed in human brain
Mark Ebbert1,2,3, Bernardo Aguzzoli-Heberle1,2, J. Anthony Brandon1,2, Kayla A. Nations1,2, Madeline Page1,2, Mark E. Wadsworth1,2, Dennis Dickson4, Peter T. Nelson1, John D. Fryer5
1Sanders-brown Center On Aging, University Of Kentucky College of Medicine, Lexington, USA, 2Department of Neuroscience, University of Kentucky College of Medicine, Lexington, USA, 3Division of Biomedical Informatics, Internal Medicine, University of Kentucky College of Medicine, Lexington, USA, 4Department of Neuroscience, Mayo Clinic, Jacksonville, USA, 5Department of Neuroscience, Mayo Clinic, Scottsdale, USA
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Contraction of brain pericytes in response to Islet Amyloid PolyPeptide is reversed by pramlintide
Cristina Nunez-diaz1, Dovilė Pocevičiūtė1, Nina Schultz1, B The Netherlands Brain Bank N B2, Karl Swärd3,
Malin Wennström1
1Clinical Memory Research Unit, Department of Clinical Sciences Malmö,Lund University, Malmö, Sweden, 2Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA, Amsterdam, Netherlands, 3Department of Experimental Medical Science, BMC D12, SE- 22184, Lund, Sweden
 Background: The islet amyloid polypeptide (IAPP), a pancreas-produced peptide, is beneficial in its monomeric form. However, IAPP aggregates, related to type 2 diabetes, are toxic both for the pancreas and brain. In the latter, IAPP is often found in vessels, where it is highly toxic for pericytes, contractile cells that regulate capillary blood flow. The aim of this study is to assess whether IAPP oligomers alter the morphology/ contractility of pericytes using an in vitro brain vasculature model and evaluate the association between IAPP and capillary contraction in human brain tissue.
Materials and Methods: Human brain pericytes were co-cultured with human cerebral microvascular endothelial cells on an extracellular matrix, forming capillary-like structures (CLS). The CLS were stimulated with oligomeric IAPP (oIAPP), sphingosine-1-phosphate (S1P), a ROCK- inhibitor (Y27632), a myosin inhibitor (blebbistatin), an IAPP receptor agonist (pramlintide), and an IAPP receptor antagonist (AC187). The proportion of round pericytes per CLS was quantified with ImageJ. Cell death was evaluated with trypan blue staining. Laminin immunostainings were performed in the hippocampus of individuals with low/high levels of total IAPP, and capillary diameters were quantified.
Results: oIAPP increased the proportion of round pericytes per CLS, while pericyte death was not significantly higher. S1P stimulation increased, while Y27632 decreased, the proportion of round pericytes. The inhibition of the IAPP receptor with AC187 only reverted IAPP effects partially, and pramlintide alone did not affect the proportion of round pericytes. The effect of oIAPP on pericytes was reverted by pramlintide, Y27632, and blebbistatin. Hippocampal capillary diameter was significantly lower in individuals with high levels of IAPP in the brain.
Background: RNASeq is typically performed using short-read sequencing technologies that, by nature, collapse all RNA isoforms for a given gene into a single expression measurement—a major oversimplification of the underlying biology. Collapsing all RNA isoforms for a single gene severely limits our ability to characterize all RNA isoforms and determine their individual functions. For example, top Alzheimer’s disease genes average ~12 distinct RNA isoforms, but we understand little about them. While heuristics to assemble short reads into full transcripts exist, these methods are inherently inaccurate. Long-read sequencing, however, can sequence entire RNA molecules, allowing researchers to accurately quantify expression for the complete set of RNA species, including de novo RNA isoforms. Here we sequenced post-mortem human brain tissue with long-reads and aligned them to the first telomere-to-telomere completed human reference genome (CHM13) to identify and quantify new gene bodies and RNA isoforms. Methods: We sequenced pre-frontal cortex tissue from five post-mortem human brain samples using Oxford Nanopore Technologies long-read sequencing (cDNA). Reads were aligned to CHM13 (minimap2), and transcripts were quantified using Bambu.
Results: We discovered 202 new, high-confidence gene bodies having five or more reads in at least three samples. We also found 622 high- confidence new RNA isoforms in known gene bodies, of which 66 are from medically relevant genes. We identified 585 medically-relevant genes expressing 2+ isoforms with 100+ reads (each) across all five samples, including four APP isoforms, four SNCA isoforms, and three MAPT isoforms.
Conclusions: Our results suggest long-reads combined with the completed CHM13 human reference genome has the potential to reveal exciting new biology relevant to human health and disease, including new gene bodies and RNA isoforms that are overlooked with standard approaches. These methods can provide a more complete picture of the transcriptomic landscape of the human brain, with potential for disease-relevant discoveries.
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