Adam Kassan

Distinct pathways of cholesterol biosynthesis impact on insulin secretion

Results from previous investigations have indicated that glucose-stimulated insulin secretion (GSIS) is affected by changes in cholesterol and its intermediates, but the precise link between secretion and cholesterol has not been thoroughly investigated. In this study, we show the contribution of both protein isoprenylation and cholesterol-dependent plasma membrane structural integrity to insulin secretion in INS-1E cells and mouse islets. Acute (2 h) inhibition of hydroxyl-methylglutaryl-CoA reductase by simvastatin (SIM) resulted in inhibition of GSIS without reduction in total cellular cholesterol content. This effect was prevented by cell loading with the isoprenyl molecule geranylgeranyl pyrophosphate. Chronic (24 h) inhibition of cholesterol biosynthesis resulted in inhibition of GSIS with a significant reduction in total cellular cholesterol content, which was also observed after the inhibition of cholesterol biosynthesis downstream of isoprenoid formation. Electron paramagnetic resonance analyses of INS-1E cells showed that the SIM-induced reduction in cholesterol increased plasma membrane fluidity. Thus, the blockade of cholesterol biosynthesis resulted in the reduction of availability of isoprenoids, followed by a reduction in the total cholesterol content associated with an increase in plasma membrane fluidity. Herein, we show the different contributions of cholesterol biosynthesis to GSIS, and propose that isoprenoid molecules and cholesterol-dependent signaling are dual regulators of proper β-cell function.

Caveolin-1 regulation of disrupted-in-schizophrenia-1 as a potential therapeutic target for schizophrenia

Schizophrenia is a debilitating psychiatric disorder manifested in early adulthood. Disrupted-in-schizophrenia-1 (DISC1) is a susceptible gene for schizophrenia (Hodgkinson et al. 2004; Millar et al. 2000; St Clair et al. 1990) implicated in neuronal development, brain maturation, and neuroplasticity (Brandon and Sawa 2011; Chubb et al. 2008). Therefore, DISC1 is a promising candidate gene for schizophrenia, but the molecular mechanisms underlying its role in the pathogenesis of the disease are still poorly understood. Interestingly, caveolin-1 (Cav-1), a cholesterol binding and scaffolding protein, regulates neuronal signal transduction and promotes neuroplasticity. In this study we examined the role of Cav-1 in mediating DISC1 expression in neurons in vitro and the hippocampus in vivo. Overexpressing Cav-1 specifically in neurons using a neuron-specific synapsin promoter (SynCav1) increased expression of DISC1 and proteins involved in synaptic plasticity (PSD95, synaptobrevin, synaptophysin, neurexin, and syntaxin 1). Similarly, SynCav1-transfected differentiated human neurons derived from induced pluripotent stem cells (hiPSCs) exhibited increased expression of DISC1 and markers of synaptic plasticity. Conversely, hippocampi from Cav-1 knockout (KO) exhibited decreased expression of DISC1 and proteins involved in synaptic plasticity. Finally, SynCav1 delivery to the hippocampus of Cav-1 KO mice and Cav-1 KO neurons in culture restored expression of DISC1 and markers of synaptic plasticity. Furthermore, we found that Cav-1 coimmunoprecipitated with DISC1 in brain tissue. These findings suggest an important role by which neuron-targeted Cav-1 regulates DISC1 neurobiology with implications for synaptic plasticity. Therefore, SynCav1 might be a potential therapeutic target for restoring neuronal function in schizophrenia. NEW & NOTEWORTHY The present study is the first to demonstrate that caveolin-1 can regulate DISC1 expression in neuronal models. Furthermore, the findings are consistent across three separate neuronal models that include rodent neurons (in vitro and in vivo) and human differentiated neurons derived from induced pluripotent stem cells. These findings justify further investigation regarding the modulatory role by caveolin on synaptic function and as a potential therapeutic target for the treatment of schizophrenia.

Caveolin-3 plays a critical role in autophagy after ischemia reperfusion.

Autophagy is a dynamic recycling process responsible for the breakdown of misfolded proteins and damaged organelles, providing nutrients and energy for cellular renovation and homeostasis. Loss of autophagy is associated with cardiovascular diseases. Caveolin-3 (Cav-3), a muscle-specific isoform, is a structural protein within caveolae and is critical to stress adaptation in the heart. Whether Cav-3 plays a role in regulating autophagy to modulate cardiac stress responses remains unknown. In the present study, we used HL-1 cells, a cardiac muscle cell line, with stable Cav-3 knockdown (Cav-3 KD) and Cav-3 overexpression (Cav-3 OE) to study the impact of Cav-3 in regulation of autophagy. We show that traditional stimulators of autophagy (i.e., rapamycin and starvation) result in upregulation of the process in Cav-3 OE cells while Cav-3 KD cells have a blunted response. Cav-3 coimmunoprecipitated with beclin-1 and Atg12, showing an interaction of caveolin with autophagy-related proteins. In the heart, autophagy may be a major regulator of protection from ischemic stress. We found that Cav-3 KD cells have a decreased expression of autophagy markers [beclin-1, light chain (LC3-II)] after simulated ischemia and ischemia-reperfusion (I/R) compared with WT, whereas OE cells showed increased expression. Moreover, Cav-3 KD cells showed increased cell death and higher level of apoptotic proteins (cleaved caspase-3 and cytochrome c) with suppressed mitochondrial function in response to simulated ischemia and I/R, whereas Cav-3 OE cells were protected and had preserved mitochondrial function. Taken together, these results indicate that autophagy regulates adaptation to cardiac stress in a Cav-3-dependent manner.

Sirtuin1 protects endothelial Caveolin-1 expression and preserves endothelial function via suppressing miR-204 and endoplasmic reticulum stress

Sirtuin1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including endothelial function. Caveolin1 (Cav1) is also an important determinant of endothelial function. We asked if Sirt1 governs endothelial Cav1 and endothelial function by regulating miR-204 expression and endoplasmic reticulum (ER) stress. Knockdown of Sirt1 in endothelial cells, and in vivo deletion of endothelial Sirt1, induced endothelial ER stress and miR-204 expression, reduced Cav1, and impaired endothelium-dependent vasorelaxation. All of these effects were reversed by a miR-204 inhibitor (miR-204 I) or with overexpression of Cav1. A miR-204 mimic (miR-204 M) decreased Cav1 in endothelial cells. In addition, high-fat diet (HFD) feeding induced vascular miR-204 and reduced endothelial Cav1. MiR-204-I protected against HFD-induced downregulation of endothelial Cav1. Moreover, pharmacologic induction of ER stress with tunicamycin downregulated endothelial Cav1 and impaired endothelium-dependent vasorelaxation that was rescued by overexpressing Cav1. In conclusion, Sirt1 preserves Cav1-dependent endothelial function by mitigating miR-204-mediated vascular ER stress.

Inhibition of p75 neurotrophin receptor does not rescue cognitive impairment in adulthood after isoflurane exposure in neonatal mice

Background Isoflurane is widely used for anaesthesia in humans. Isoflurane exposure of rodents prior to post-natal day 7 (PND7) leads to widespread neurodegeneration in laboratory animals. Previous data from our laboratory suggest an attenuation of apoptosis with the p75 neurotrophin receptor (p75NTR) inhibitor TAT-Pep5. We hypothesized that isoflurane toxicity leads to behavioural and cognitive abnormalities and can be rescued with pre-anaesthesia administration of TAT-Pep5. Methods Neonatal mouse pups were pretreated with either TAT-Pep5 (25 μl, 10 μM i.p.) or a scrambled control peptide (TAT-ctrl; 25 μl, 10 μM i.p.) prior to isoflurane exposure (1.4%; 4 h) or control ( n  = 15-26/group). Three to 5 months after exposure, behavioural testing and endpoint assays [brain volume (stereology) and immunoblotting] were performed. Results No significant difference was observed in open field, T-maze, balance beam or wire-hanging testing. The Barnes maze revealed a significant effect of isoflurane ( P  = 0.019) in errors to find the escape tunnel during the day 5 probe trial, a finding indicative of impaired short-term spatial memory. No difference was found for brain volumes or protein expression. TAT-Pep5 treatment did not reverse the effects of isoflurane on neurocognitive behaviour. Conclusion A single isoflurane exposure to early post-natal mice caused a hippocampal-dependent memory deficit that was not prevented by pre-administration of TAT-Pep5, although TAT-Pep5, an inhibitor of p75NTR, has been shown to reduce isoflurane-induced apoptosis. These findings suggest that neuronal apoptosis is not requisite for the development of cognitive deficits in the adults attendant with neonatal anaesthetic exposure.

Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes

Statins, which reduce LDL‐cholesterol by inhibition of 3‐hydroxy‐3‐methylglutaryl–coenzyme A reductase, are among the most widely prescribed drugs. Skeletal myopathy is a known statin‐induced adverse effect associated with mitochondrial changes. We hypothesized that similar effects would occur in cardiac myocytes in a lipophilicity‐dependent manner between 2 common statins: atorvastatin (lipophilic) and pravastatin (hydrophilic). Neonatal cardiac ventricular myocytes were treated with atorvastatin and pravastatin for 48 h. Both statins induced endoplasmic reticular (ER) stress, but only atorvastatin inhibited ERK1/2T202/Y204, AktSer473, and mammalian target of rapamycin signaling; reduced protein abundance of caveolin‐1, dystrophin, epidermal growth factor receptor, and insulin receptor‐β; decreased Ras homolog gene family member A activation; and induced apoptosis. In cardiomyocyte‐equivalent HL‐1 cells, atorvastatin, but not pravastatin, reduced mitochondrial oxygen consumption. When male mice underwent atorvastatin and pravastatin administration per os for up to 7 mo, only long‐term atorvastatin, but not pravastatin, induced elevated serum creatine kinase; swollen, misaligned, size‐variable, and disconnected cardiac mitochondria; alteration of ER structure; repression of mitochondria‐ and endoplasmic reticulum–related genes; and a 21% increase in mortality in cardiac‐specific vinculin‐knockout mice during the first 2 months of administration. To our knowledge, we are the first to demonstrate in vivo that long‐term atorvastatin administration alters cardiac ultrastructure, a finding with important clinical implications.——Godoy, J. C., Niesman, I. R., Busija, A.R., Kassan, A., Schilling, J. M., Schwarz, A., Alvarez, E. A., Dalton, N. D., Drummond, J. C., Roth, D.M., Kararigas, G., Patel, H. H., Zemljic‐Harpf, A. E. Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes. FASEB J. 33, 1209–1225 (2019). www.fasebj.org