Biology subjects

Pfenning, A. R.

Publications and source records attributed to Pfenning, A. R..

29 records · Page 2Linked to original sources

bioRxiv2021

Neurons burdened by DNA double strand breaks incite microglia activation through antiviral-like signaling in neurodegeneration.

DNA double strand breaks (DSBs) are linked to aging, neurodegeneration, and senescence1,2. However, the role played by neurons burdened with DSBs in disease-associated neuroinflammation is not well understood. Here, we isolate neurons harboring DSBs from the CK-p25 mouse model of neurodegeneration through fluorescence-activated nuclei sorting (FANS), and characterize their transcriptomes using single-nucleus, bulk, and spatial sequencing techniques. We find that neurons harboring DSBs enter a late-stage DNA damage response marked by the activation of senescent and antiviral-like immune pathways. We identify the NFkB transcription factor as a master regulator of immune gene expression in DSB-bearing neurons, and find that the expression of cytokines like Cxcl10 and Ccl2 develop in DSB-bearing neurons before glial cell types. Alzheimers Disease pathology is significantly associated with immune activation in excitatory neurons, and direct purification of DSB-bearing neurons from Alzheimers Disease brain tissue further validates immune gene upregulation. Spatial transcriptomics reveal that regions of brain tissue dense with DSB-bearing neurons also harbor signatures of inflammatory microglia, which is ameliorated by NFkB knock down in neurons. Inhibition of NFkB or depletion of Ccl2 and Cxcl10 in DSB-bearing neurons also reduces microglial activation in organotypic brain slice culture. In conclusion, we find that in the context of age-associated neurodegenerative disease, DSBs activate immune pathways in neurons, which in turn adopt a senescence associated secretory phenotype to elicit microglia activation. These findings highlight a novel role for neurons in the mechanism of age-associated neuroinflammation. SummaryIt is unclear how age-associated DNA double strand break (DSB) accumulation in neurons influences the progression of cellular senescence and neurodegenerative disease. Here, we leverage mouse models of neurodegeneration, single-nucleus, bulk, and spatial transcriptomics from Alzheimers disease patients, mouse models, and primary neuron cultures to dissect the immune signaling pathways initiated by DSB-bearing neurons that trigger neuroinflammation.

neuroscience↗

bioRxiv2021

Molecular rhythm alterations in prefrontal cortex and nucleus accumbens associated with opioid use disorder

Severe and persistent disruptions to sleep and circadian rhythms are common features of people with opioid use disorder (OUD). Preclinical findings suggest altered molecular rhythms in the brain are involved in opioid reward and dependence. However, whether molecular rhythms are disrupted in brains of people with OUD remained an open question, critical to understanding the role of circadian rhythms in opioid addiction. We previously used subjects times of death (TOD) as a marker of time of day to investigate transcriptional rhythm alterations in psychiatric disorders. Using TOD and RNA sequencing, we discovered rhythmic transcripts in both the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), key brain areas involved in opioid addiction, were largely distinct between OUD and unaffected comparison subjects. Further, fewer rhythmic transcripts were identified in DLPFC of OUD subjects compared to unaffected subjects, but nearly double the number of rhythmic transcripts were found in the NAc of OUD subjects. In OUD, rhythmic transcripts in the NAc peaked either in the evening or near sunrise, and were associated with dopamine, opioid, and GABAergic neurotransmission. Co-expression network analysis identified several OUD-specific modules in the NAc, enriched for transcripts involved in the modulation of dopamine and GABA synapses, including glutamatergic signaling and extracellular matrices. Integrative analyses with human GWAS revealed that rhythmic transcripts in DLPFC and NAc were enriched for genomic loci associated with sleep duration and insomnia. Overall, our results connect transcriptional rhythm changes in dopamine, opioid, and GABAergic synaptic signaling in human brain to sleep-related phenotypes and OUD.

neuroscience↗

bioRxiv2021

Machine learning sequence prioritization for cell type-specific enhancer design

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations, enabling characterization of their roles in behavior and in disease states. Available approaches for engineering targeted technologies for new neuron subtypes are low-yield, involving intensive transgenic strain or virus screening. Here, we introduce SNAIL (Specific Nuclear-Anchored Independent Labeling), a new virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and using them to make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV) neurons. Furthermore, we show that nuclear isolation using SNAIL in wild type mice is sufficient to capture characteristic open chromatin features of PV neurons in the cortex, striatum, and external globus pallidus. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

genomics↗

bioRxiv2020

Predicting lineage-specific differences in open chromatin across dozens of mammalian genomes

BackgroundEvolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation at many enhancers, distal regulatory elements that control spatio-temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific, code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high. ResultsWe present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that is able to predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Then, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity and liver enhancers associated with liver regeneration have a stronger tendency than the general population to have predicted lineage-specific open chromatin. ConclusionThe framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements.

genetics↗

bioRxiv2020

The Regulatory Evolution of the Primate Fine-Motor System

In mammals, fine motor control is essential for skilled behavior, and is subserved by specialized subdivisions of the primary motor cortex (M1) and other components of the brains motor circuitry. We profiled the epigenomic state of several components of the Rhesus macaque motor system, including subdivisions of M1 corresponding to hand and orofacial control. We compared this to open chromatin data from M1 in rat, mouse, and human. We found broad similarities as well as unique specializations in open chromatin regions (OCRs) between M1 subdivisions and other brain regions, as well as species- and lineage-specific differences reflecting their evolutionary histories. By distinguishing shared mammalian M1 OCRs from primate- and human-specific specializations, we highlight gene regulatory programs that could subserve the evolution of skilled motor behaviors such as speech and tool use.

neuroscience↗

bioRxiv2020

Transcriptional Diversity of Medium Spiny Neurons in the Primate Striatum

The striatum is the interface between dopamine reward signals and cortico-basal ganglia circuits that mediate diverse behavioral functions. Medium spiny neurons (MSNs) constitute the vast majority of striatal neurons and are traditionally classified as direct- or indirect-pathway neurons. However, that traditional model does not explain the anatomical and functional diversity of MSNs. Here, we defined molecularly distinct MSN types in the primate striatum, including (1) dorsal striatum MSN types associated with striosome and matrix compartments, (2) ventral striatum types associated with the nucleus accumbens shell and olfactory tubercle, and (3) an MSN-like type restricted to -opioid receptor rich islands in the ventral striatum. These results lay the foundation for achieving cell type-specific transgenesis in the primate striatum and provide a blueprint for investigating circuit-specific processing.

neuroscience↗

bioRxiv2020

scAAVengr: Single-cell transcriptome-based quantification of engineered AAVs in non-human primate retina

Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to quantify efficiency of AAV-mediated gene expression across all cell types. scAAVengr allows for definitive, head-to-head comparison of vectors in the same animal. To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. These results validate scAAVengr as a powerful method for development of AAV vectors.

bioengineering↗

bioRxiv2020

Addiction-associated genetic variants implicate brain cell type- and region-specific cis-regulatory elements in addiction neurobiology

Recent large genome-wide association studies (GWAS) have identified multiple confident risk loci linked to addiction-associated behavioral traits. Genetic variants linked to addiction-associated traits lie largely in non-coding regions of the genome, likely disrupting cis-regulatory element (CRE) function. CREs tend to be highly cell type-specific and may contribute to the functional development of the neural circuits underlying addiction. Yet, a systematic approach for predicting the impact of risk variants on the CREs of specific cell populations is lacking. To dissect the cell types and brain regions underlying addiction-associated traits, we applied LD score regression to compare GWAS to genomic regions collected from human and mouse assays for open chromatin, which is associated with CRE activity. We found enrichment of addiction-associated variants in putative CREs marked by open chromatin in neuronal (NeuN+) nuclei collected from multiple prefrontal cortical areas and striatal regions known to play major roles in reward and addiction. To further dissect the cell type-specific basis of addiction-associated traits, we also identified enrichments in human orthologs of open chromatin regions of mouse neuronal subtypes: cortical excitatory, D1, D2, and PV. Lastly, we developed machine learning models from mouse cell type-specific regions of open chromatin to further dissect human NeuN+ open chromatin regions into cortical excitatory or striatal D1 and D2 neurons and predict the functional impact of addiction-associated genetic variants. Our results suggest that different neuronal subtypes within the reward system play distinct roles in the variety of traits that contribute to addiction. Significance StatementWe combine statistical genetic and machine learning techniques to find that the predisposition to for nicotine, alcohol, and cannabis use behaviors can be partially explained by genetic variants in conserved regulatory elements within specific brain regions and neuronal subtypes of the reward system. This computational framework can flexibly integrate open chromatin data across species to screen for putative causal variants in a cell type-and tissue-specific manner across numerous complex traits.

genomics↗

bioRxiv2020

Broad Host Range of SARS-CoV-2 Predicted by Comparative and Structural Analysis of ACE2 in Vertebrates

The novel coronavirus SARS-CoV-2 is the cause of Coronavirus Disease-2019 (COVID-19). The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of 410 vertebrates, including 252 mammals, to study cross-species conservation of ACE2 and its likelihood to function as a SARS-CoV-2 receptor. We designed a five-category ranking score based on the conservation properties of 25 amino acids important for the binding between receptor and virus, classifying all species from very high to very low. Only mammals fell into the medium to very high categories, and only catarrhine primates in the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 binding, and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (<0.1%) variants in 10/25 binding sites. In addition, we observed evidence of positive selection in ACE2 in multiple species, including bats. Utilized appropriately, our results may lead to the identification of intermediate host species for SARS-CoV-2, justify the selection of animal models of COVID-19, and assist the conservation of animals both in native habitats and in human care.

genomics↗

bioRxiv2019

Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals

All living things experience entropy, manifested as a loss of inherited genetic and epigenetic information over time. As budding yeast cells age, epigenetic changes result in a loss of cell identity and sterility, both hallmarks of yeast aging. In mammals, epigenetic information is also lost over time, but what causes it to be lost and whether it is a cause or a consequence of aging is not known. Here we show that the transient induction of genomic instability, in the form of a low number of non-mutagenic DNA breaks, accelerates many of the chromatin and tissue changes seen during aging, including the erosion of the epigenetic landscape, a loss of cellular identity, advancement of the DNA methylation clock and cellular senescence. These data support a model in which a loss of epigenetic information is a cause of aging in mammals.\n\nOne Sentence SummaryThe act of repairing DNA breaks induces chromatin reorganization and a loss of cell identity that may contribute to mammalian aging

molecular biology↗

bioRxiv2018

Epigenome-wide study uncovers tau pathology-driven changes of chromatin organization in the aging human brain

Accumulation of tau and amyloid-{beta} are two pathologic hallmarks of Alzheimers disease (AD). Here, we conducted an epigenome-wide association study using the H3K9 acetylation (H3K9Ac) mark in 669 aged human prefrontal cortices: in contrast to amyloid-{beta}, tau protein burden had a broad effect on the epigenome, affecting 5,590 out of 26,384 H3K9Ac domains. Tau-related alterations aggregated in large genomic segments reflecting spatial chromatin organization, and the magnitude of these effects correlated with the segments nuclear lamina association. We confirmed the functional relevance of these chromatin changes by demonstrating (1) consistent transcriptional changes in three independent datasets and (2) similar findings in two AD mouse models. Finally, we found that tau overexpression in iPSC-derived neurons disrupted chromatin organization and that these effects could be blocked by a small molecule predicted to reverse the tau effect. Thus, we report large-scale tau-driven chromatin rearrangements in the aging human brain that may be reversible with HSP90 inhibitors.

neuroscience↗