Ciclesonide activates glucocorticoid signaling in neonatal rat lung but does not trigger adverse effects in the cortex and cerebellum

Synthetic glucocorticoids (sGCs) such as dexamethasone (DEX), while used to mitigate inflammation and disease progression in premature infants with severe bronchopulmonary dysplasia (BPD), are also associated with significant adverse neurologic effects such as reductions in myelination and abnormalities in neuroanatomical development. Ciclesonide (CIC) is a sGC prodrug approved for asthma treatment that exhibits limited systemic side effects. Carboxylesterases enriched in the lower airways convert CIC to the glucocorticoid receptor (GR) agonist des-CIC. We therefore examined whether CIC would likewise activate GR in neonatal lung but have limited adverse extra-pulmonary effects, particularly in the developing brain. Neonatal rats were administered subcutaneous injections of CIC, DEX or vehicle from postnatal days 1–5 (PND1-PND5). Systemic effects linked to DEX exposure, including reduced body and brain weight, were not observed in CIC treated neonates. Furthermore, CIC did not trigger the long-lasting reduction in myelin basic protein expression in the cerebral cortex nor cerebellar size caused by neonatal DEX exposure. Conversely, DEX and CIC were both effective at inducing the expression of select GR target genes in neonatal lung, including those implicated in lung-protective and anti-inflammatory effects. Thus, CIC is a promising, novel candidate drug to treat or prevent BPD in neonates given its activation of GR in neonatal lung and limited adverse neurodevelopmental effects. Furthermore, since sGCs such as DEX administered to pregnant women in pre-term labor can adversely affect fetal brain development, the neurological-sparing properties of CIC, make it an attractive alternative for DEX to treat pregnant women severely ill with respiratory illness, such as with asthma exacerbations or COVID-19 infections

Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms

<div><p>Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate <i>in vitro</i>. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.</p></div

Pravastatin decreases NSPC expansion <i>in vitro</i>.

<p>(A) Representative images of neurosphere cultures. Treatments include vehicle, 1 μM and 25 μM pravastatin (prava) on days 1, 3 and 5 after treatment. Bright field images were taken on an inverted microscope at 10x magnification. (B) Quantification was performed on images taken in triplicate using image J software on days 0, 1, 3 and 5 after treatment with vehicle, 1 μM or 25 μM prava (n = 5). (C) After imaging, NSPCs were collected for automated cell counting in duplicate (n = 5).</p

Pravastatin induces CBP gene expression in NSPCs.

<p>(A) Ingenuity pathway gene ontology analysis identified “Superpathway of Cholesterol Biosynthesis” and related pathways as the top regulated pathways in neurospheres after a 10 μM prava treatment for 24 hours.–LOG10 (p-value) and percentage overlap with identified pathways are reported (n = 6). (B) Ingenuity pathway gene ontology analysis identified “Cholesterol Biosynthesis” as the top toxicity function in neurosphere after 10 μM prava treatment for 24 hours (n = 6). (C) Average mRNA expression of cholesterol pathway and cholesterol metabolism genes in NSPCs after treatment with vehicle, 1 μM or 25 μM prava for 24 hours (n = 3–5). Expression was normalized to <i>GAPDH</i> and the vehicle treatment group for each gene. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to controls, unless otherwise demonstrated with a horizontal bracket).</p

Pravastatin activates SREBP2 in NSPCs.

<p>(A) Ingenuity pathway analysis identified top upstream regulators of transcriptional change induced by 10 μM prava. (B) Network of transcriptional regulation by the SCAP-INSIG-SREBP2 complex. (C) Representative western blot of cleaved SREBP2 (cSREBP2) from NSPCs treated with vehicle, 1 μM or 25 μM prava for 24 hours. The treatment group of each band matches the column of the histogram directly below. (D) Average intensity of bands corresponding to 24 hour treatment with vehicle, 1 μM or 25 μM prava for cSREBP2 normalized to ß-Actin (n = 7). (*p<0.05, **p<0.01, ****p<0.0001 compared to controls, unless otherwise demonstrated with a horizontal bracket).</p

Statins alter mRNA expression in differentiating NSPCs through a non-CBP dependent mechanism.

<p>Average mRNA expression of the neuronal markers (A) <i>Map2</i> (n = 6), (B) Tuj1 (n = 6), astrocyte marker (C) GFAP (n = 6), and oligodendrocyte marker (D) Olig2 (n = 6) after four days of NSPC differentiation in 10 uM prava. Average mRNA expression of the neuronal markers (E) <i>Map2</i> (n = 6) and (F) <i>Tuj1</i> (n = 6) as well as the astrocyte marker (G) <i>GFAP</i> (n = 6) and oligodendrocyte marker (H) <i>Olig2</i> (n = 6) after four days of NSPC differentiation in 1uM simva. (I) Average mRNA expression of <i>GFAP</i> in cells after four days of differentiation in 10uM prava co-treated with 1 uM or 10 uM FPP (n = 5). (J) Average mRNA expression of <i>Ldlr</i> in cells after four days of differentiation in 10 uM prava co-treated with 1 uM or 10 uM FPP (n = 5). (*p<0.05, **p<0.01, ***p<0.001 compared to vehicle unless otherwise indicated by brackets).</p

FPP prevents pravastatin dependent SREBP2 activation and induction of cell death.

<p>(A) Representative western blot of cleaved SREBP2 (cSREBP2) from NSPCs treated with vehicle, 10 μM prava, 1 μM farnesyl pyrophosphate (FPP), 10 μM FPP, 10 μM prava and 1 μM FPP, or 10 μM prava and 10 μM FPP, respectively, for 24 hours. The treatment group of each band matches the column of the histogram directly below. (B) Average intensity of cSREBP2 bands normalized to ß-actin (n = 7). (C) Average mRNA expression of <i>Ldlr</i>. Expression was normalized to <i>GAPDH</i> (n = 5). (D) Representative western blot of cleaved PARP (cPARP). The treatment group of each band matches the column of the histogram directly below. (E) Average intensity of cPARP bands normalized to ß-actin (n = 7) (***p<0.001, ****p<0.0001 compared to vehicle; #p<0.05, ##p<0.01, ###p<0.001 compared to 10 μM prava).</p

Pravastatin increases cyclin D1 expression but prevents passage through S-Phase in NSPCs.

<p>A. Average expression of <i>Ccnd1</i> mRNA as assessed by qRT-PCR (n = 5). B. Representative western blot of cyclin D1 from neurospheres treated with vehicle, 1 uM or 25 uM prava for 24 hours C. Average intensity of bands corresponding to 24 hour treatment with vehicle, 1 uM or 25 uM prava for cyclin D1 normalized to ß-Actin (n = 5). D. Percentage of EdU+ NSPCs treated with 1 uM or 25 uM prava for three days with EdU incorporation during the final 4 hours, quantified by flow cytometry (n = 8). E. Representative image of dissociated neurospheres after three days treatment with vehicle for DAPI and EdU incorporation. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to vehicle, unless otherwise indicated by a horizontal bracket).</p

Pravastatin enhances cell death and reduces G1 accumulation of NSPCs.

<p>A. Quantification of NSPCs in sub-G1, G0/G1 and S/G2/M. Diva software was used to count the number of NSPCs that were identified as sub-G1, in G0/G1 or in S/G2/M phase. The histogram represents the mean percentage of NSPCs and standard error of the mean (SEM) in each category after treatment with vehicle, 1 uM or 25 uM prava (n = 5). B. Representative western blot of anti-cleaved PARP (cPARP) from neurospheres treated with vehicle, 1 uM or 25 uM pravastatin (prava) for 24 hours. C. Average intensity of bands corresponding to 24 hour treatment with vehicle, 1 uM or 25 uM prava for cleaved PARP normalized to ß-Actin (n = 5). D. Average intensity of bands corresponding to 24 hour treatment with vehicle, 1 uM or 25 uM prava for cleaved caspase normalized to alpha-tubulin (n = 3). (**p<0.01, ***p<0.001, compared to vehicle).</p