Biology subjects

Meyerholz, D. K.

Publications and source records attributed to Meyerholz, D. K..

21 records · Page 2Linked to original sources

bioRxiv2020

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract

BackgroundZoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. Materials and methodsWe systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. FindingsOur results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19. InterpretationThese results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrevious studies of ACE2 mRNA transcript abundance in the human respiratory tract have suggested a possible association between ACE2 expression and age, sex, and the presence of comorbidities. However, these studies have provided conflicting results, as well as a lack of protein validation. Previous ACE2 protein studies have been limited by a paucity of lung tissue samples and reports that have produced contradictory results. Added value of this studyUsing a combination of single-cell RNA sequencing and immunohistochemistry, we describe ACE2 expression in the human respiratory tract. Staining protocols were optimized and validated to show consistent apical localization and avoid non-specific staining. We show ACE2 protein is found in subsets of airway cells and is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development for COVID-19, respectively. We show age, sex, and comorbidities do not increase ACE2 protein expression in the human respiratory tract. Implications of all the available evidenceACE2 protein abundance does not correlate with risk factors for severe clinical outcomes, but in some cases showed an inversed relationship. Features driving COVID-19 susceptibility and severity are complex, our data suggests factors other than ACE2 protein abundance as important determinants of clinical outcomes.

cell biology↗

bioRxiv2019

Validating indicators of CNS disorders in a swine model of neurological disease

Genetically modified porcine disease models are becoming increasingly important for studying molecular, physiological and pathological characteristics of human disorders. Given their limited history, there remains a great need for proven reagents in swine tissue. To provide a resource for neurological models of disease, we validated antibodies by immunohistochemistry for use in examining central nervous system (CNS) markers. To validate these tools in a relevant model, we utilized a recently developed miniswine model of neurofibromatosis type 1 (NF1). NF1 is a tumor predisposition disorder, presenting with different type of tumors. Additionally, neurological associated symptomologies may include chronic pain, cognitive impairment, and behavioral abnormalities, making this miniswine model an ideal candidate for validating CNS-relevant antibodies. We validate antibodies implicated in glial inflammation (CD68), oligodendrocyte development (NG2, O4, Olig2, and myelin PLP), and neuron differentiation and neurotransmission (doublecortin, GAD67, and tyrosine hydroxylase) by examining cellular localization and brain region specificity. Additionally, we confirm the utility of anti-GFAP, anti-Iba1, and anti-MBP antibodies, previously validated in swine, by testing their immunoreactivity across multiple brain regions in mutant NF1 samples. These validated immunostaining protocols for CNS markers provide a useful resource, furthering the utility of the genetically modified miniswine for translational and clinical applications.

neuroscience↗

bioRxiv2018

Sex-dependent differences in pain and sleep in a porcine model of Neurofibromatosis type 1

Neurofibromatosis Type 1 (NF1) is an autosomal dominant genetic disorder resulting from germline mutations in the NF1 gene, which encodes neurofibromin. Patients experience a variety of symptoms, but pain in the context of NF1 remains largely underrecognized. Here, we characterize nociceptive signaling and pain behaviors in a miniswine harboring a disruptive NF1 mutation (exon 42 deletion). We explore these phenotypes in relationship to collapsin response mediator protein 2 (CRMP2), a known interactor of neurofibromin. Mechanistically, we found two previously unknown phosphorylated residues of CRMP2 in NF1+/ex42del pig dorsal root ganglia (DRGs) and replicated increased voltage-gated calcium channel currents in NF1+/ex42del pig DRGs previously described in rodent models of NF1. We present the first characterization of pain-related behaviors in a pig model of NF1, identifying unchanged agitation scores, lower tactile thresholds (allodynia), and decreased response latencies to thermal laser stimulation (hyperalgesia) in the NF1 mutant animals; NF1+/ex42del pigs demonstrated sexually dimorphic behaviors. NF1+/ex42del pigs showed reduced sleep quality and increased resting, two health-related quality of life symptoms found to be comorbid in people with NF1 pain. Finally, we show decreased depolarization-evoked calcium influx in both wildtype and NF1+/ex42del pig DRGs treated with CRMP2 phosphorylation inhibitor (5)-lacosamide. Our data supports use of NF1+/ex42del pigs as an ideal model for studying NF1-associated pain and are a better model for understanding the pathophysiology of NF1 compared to rodents. Moreover, our findings demonstrate that interfering with CRMP2 phosphorylation might be a promising therapeutic strategy for NF1-related pain management.

neuroscience↗