Many lung diseases result from a failure of efficient regeneration of damaged alveolar epithelial cells (AECs) after lung injury. During regeneration, AEC2s proliferate to replace lost cells, after which proliferation halts and some AEC2s transdifferentiate into AEC1s to restore normal alveolar structure and function. Although the mechanisms underlying AEC2 proliferation have been studied, the mechanisms responsible for halting proliferation and inducing transdifferentiation are poorly understood. To identify candidate signaling pathways responsible for halting proliferation and inducing transdifferentiation, we performed single cell RNA sequencing on AEC2s during regeneration in a murine model of lung injury induced by intratracheal LPS. Unsupervised clustering revealed distinct subpopulations of regenerating AEC2s: proliferating, cell cycle arrest, and transdifferentiating. Gene expression analysis of these transitional subpopulations revealed that TGFβ signaling was highly upregulated in the cell cycle arrest subpopulation and relatively downregulated in transdifferentiating cells. In cultured AEC2s, TGFβ was necessary for cell cycle arrest but impeded transdifferentiation. We conclude that during regeneration after LPS-induced lung injury, TGFβ is a critical signal halting AEC2 proliferation but must be inactivated to allow transdifferentiation. This study provides insight into the molecular mechanisms regulating alveolar regeneration and the pathogenesis of diseases resulting from a failure of regeneration.

Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a proinflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from induced pluripotent stem cell (iPSC) lines from patients with primary progressive multiple sclerosis (PPMS) failed to promote oligodendrocyte progenitor cell (OPC) maturation, whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+progenitor cells within white matter lesions of human progressive MS (PMS) autopsy brain tissues and iPS-derived NPCs from patients with PPMS. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin, which then enhanced PPMS NPC support for oligodendrocyte (OL) differentiation. A proteomic analysis of the PPMS NPC secretome identified high-mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of OL differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in PMS, which may impact how this disease is modeled and inform development of future myelin regeneration strategies.

Abstract

BACKGROUND:

Progressive familial intrahepatic cholestasis type 3 (PFIC3) often leads to end-stage liver disease before adulthood with limited therapeutic options, due to impaired ABCB4 dependent phospholipid transport to bile. To restore ABCB4 function we propose adeno-associated virus serotype 8 (AAV8)-mediated gene therapy directed to the liver, although achieving stable transgene expression in hyperproliferative tissue is challenging. By restoring the phospholipid content in bile to levels that prevent liver damage, this study aims for stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3.

METHODS:

Ten weeks old Abcb4-/- mice received a single dose of AAV8-hABCB4 (n=10) or AAV8-GFP (n=7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by (immuno-)histochemistry.

RESULTS:

Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration, which restored biliary phospholipid excretion to levels that ameliorate liver damage. This resulted in normalization of plasma cholestatic markers, prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study.

CONCLUSION:

Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3, encouraging translational studies to verify clinical feasibility.

LAY SUMMARY:

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. Also proliferation of liver cells was reduced, which contributes to long term correction of the phenotype. Limitations of the mouse model requires further studies to evaluate if this approach can be applied in PFIC3 patients.

Proprioceptive feedback from group Ia/II muscle spindle afferents and group Ib Golgi tendon afferents is critical for the normal execution of most motor tasks, yet how these distinct proprioceptor subtypes emerge during development remains poorly understood. Using molecular genetic approaches in mice of either sex, we identified twenty-four transcripts that have not previously been associated with a proprioceptor identity. Combinatorial expression analyses of these markers reveal at least three molecularly distinct proprioceptor subtypes. In addition, we find that twelve of these transcripts are expressed well after proprioceptors innervate their respective sensory receptors, and expression of three of these markers - including the heart development molecule Heg1 - is significantly reduced in mice that lack muscle spindles. These data reveal Heg1 as a putative marker for proprioceptive muscle spindle afferents. Moreover, they suggest that the phenotypic specialization of functionally distinct proprioceptor subtypes depends, in part, on extrinsic sensory receptor organ-derived signals.Significance statement:Sensory feedback from muscle spindle (MS) and Golgi tendon organ (GTO) sensory end-organs is critical for normal motor control, but how distinct MS and GTO afferent sensory neurons emerge during development remains poorly understood. Using (bulk) transcriptome analysis of genetically identified proprioceptors, this work reveals molecular markers for distinct proprioceptor subsets, including some that appear selectively expressed in MS afferents. Detailed analysis of the expression of these transcripts provides evidence that MS/GTO afferent subtype phenotypes may, at least in part, emerge through extrinsic - sensory end-organ derived - signals.

Pervasive transcription is a feature of the human genome that requires better understanding. Over the last decade or so, RNA species longer than 200 nucleotides-dubbed long non-coding RNA (lncRNAs)-had been found in sense or anti-sense orientation within or outside of genes that encode proteins. Importantly, lncRNA-mediated gene regulation and the elements that control lncRNA expression are a source of fascination among molecular biologists. In vascular biology, a dozen or so lncRNAs had been identified, and progress occurs each day. In this review, we highlighted our laboratories' contribution to the lncRNA field by discussing lessons learned from two lncRNAs in the tyrosine kinase containing immunoglobulin and epidermal growth factor homology1 (Tie1) and delta-like 4 (Dll4) loci. These genes are responsible for basic vascular patterning and pathophysiological remodeling in angiogenesis.

We present the first documented case of Trypanosoma cruzi-induced orchitis in a rhesus macaque. Additionally, we describe an in situ hybridization-based assay to confirm T. cruzi infection in formalin-fixed tissues.

Transcription factors (TFs) are dosage-sensitive master regulators of gene expression, with haploinsufficiency frequently leading to life-threatening disease. Numerous mechanisms have evolved to tightly regulate the expression and activity of TFs at the transcriptional, translational, and posttranslational levels. A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors in the genome, but the regulatory relationship between these lncRNAs and their neighboring TFs is unclear. We identified a regulatory feedback loop between the TF Foxa2 and a downstream lncRNA, Falcor (Foxa2-adjacent long noncoding RNA). Foxa2 directly represses Falcor expression by binding to its promoter, while Falcor functions in cis to positively regulate the expression of Foxa2. In the lung, loss of Falcor is sufficient to lead to chronic inflammatory changes and defective repair after airway epithelial injury. Moreover, disruption of the Falcor-Foxa2 regulatory feedback loop leads to altered cell adhesion and migration, in turn resulting in chronic peribronchial airway inflammation and goblet cell metaplasia. These data reveal that the lncRNA Falcor functions within a regulatory feedback loop to fine-tune the expression of Foxa2, maintain airway epithelial homeostasis, and promote regeneration.

Abstract

AIMS:

Traditional serrated adenoma (TSA) is an uncommon type of colorectal serrated polyp. RSPO fusions, which potentiate WNT signaling, are common and characteristic genetic alterations in TSA. The aim of this study was to further characterize the prevalence and variation of RSPO fusions in TSA.

METHODS AND RESULTS:

Quantitative PCR analysis of 99 TSAs revealed overexpression of RSPO2 and RSPO3 in 6 and 29 lesions, respectively. Reverse-transcription PCR identified previously reported PTPRK-RSPO3 fusion transcripts in all the 29 TSAs with RSPO3 overexpression, confirming that PTPRK-RSPO3 is the predominant RSPO fusions in TSAs. Among the six lesions with RSPO2 overexpression, two overexpressed full-length RSPO2. An EIF3E-RSPO2 fusion, which is a known recurrent RSPO fusion in colorectal cancer, was detected in three lesions. In addition, rapid amplification of cDNA ends identified a novel PIEZO1-RSPO2 fusion in one TSA. All the four TSAs with RSPO2 fusions concurrently had KRAS mutations and showed the classical histological features.

CONCLUSIONS:

The present study identified EIF3E-RSPO2 and PIEZO1-RSPO2 fusions in TSAs. Our observations expand the spectrum of RSPO fusions in TSAs and suggest that TSAs are precursors of colorectal cancers with these RSPO2 fusions. 

Neuritic plaques, a pathological hallmark in Alzheimer's disease (AD) brains, comprise extracellular aggregates of amyloid-beta (Aβ) peptide and degenerating neurites that accumulate autolysosomes. We found that, in the brains of patients with AD and in AD mouse models, Aβ plaque-associated Olig2- and NG2-expressing oligodendrocyte progenitor cells (OPCs), but not astrocytes, microglia, or oligodendrocytes, exhibit a senescence-like phenotype characterized by the upregulation of p21/CDKN1A, p16/INK4/CDKN2A proteins, and senescence-associated β-galactosidase activity. Molecular interrogation of the Aβ plaque environment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Direct exposure of cultured OPCs to aggregating Aβ triggered cell senescence. Senolytic treatment of AD mice selectively removed senescent cells from the plaque environment, reduced neuroinflammation, lessened Aβ load, and ameliorated cognitive deficits. Our findings suggest a role for Aβ-induced OPC cell senescence in neuroinflammation and cognitive deficits in AD, and a potential therapeutic benefit of senolytic treatments.

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease.

By virtue of their extensive axonal arborisation and perisomatic synaptic targeting, cortical inhibitory Parvalbumin (PV) cells strongly regulate principal cell output and plasticity and modulate experience-dependent refinement of cortical circuits during development. An interesting aspect of PV cell connectivity is its prolonged maturation time course, which is completed only by end of adolescence. The p75 neurotrophin receptor (p75NTR) regulates numerous cellular functions, however its role on cortical circuit development and plasticity remains elusive, mainly because localizing p75NTR expression with cellular and temporal resolution has been challenging.By using RNAscope and a modified version of the Proximity Ligation Assay, we found that p75NTR expression in PV cells decreases between the second and fourth postnatal week, at a time when PV cell synapse numbers increase dramatically. Conditional knockout of p75NTR in single PV neurons in vitro and in PV cell networks in vivo causes precocious formation of PV cell perisomatic innervation and perineural nets around PV cell somata, therefore suggesting that p75NTR expression modulates the timing of maturation of PV cell connectivity in the adolescent cortex.Remarkably, we found that PV cells still express p75NTR in adult mouse cortex of both sexes and that its activation is sufficient to destabilize PV cell connectivity and to restore cortical plasticity following monocular deprivation in vivo. Altogether, our results show that p75NTR activation dynamically regulates PV cell connectivity, and represents a novel tool to foster brain plasticity in adults.SIGNIFICANCE STATEMENTIn the cortex, inhibitory, GABA-releasing neurons control the output and plasticity of excitatory neurons. Within this diverse group, parvalbumin-expressing (PV) cells form the larger inhibitory system. PV cell connectivity develops slowly, reaching maturity only at the end of adolescence, however the mechanisms controlling the timing of its maturation are not well understood. We discovered that the expression of the neurotrophin receptor p75NTR in PV cells inhibits the maturation of their connectivity in a cell autonomous fashion, both in vitro and in vivo and that p75NTR activation in adult PV cells promotes their remodelling and restores cortical plasticity. These results reveal a new p75NTR function in the regulation of the time course of PV cell maturation and in limiting cortical plasticity.