Lipid Dynamics at Contact Sites Between the Endoplasmic Reticulum and Other Organelles.

Phospholipids are synthesized primarily within the endoplasmic reticulum and are subsequently distributed to various subcellular membranes to maintain the unique lipid composition of specific organelles. As a result, in most cases, the steady-state localization of membrane phospholipids does not match their site of synthesis. This raises the question of how diverse lipid species reach their final membrane destinations and what molecular processes provide the energy to maintain the lipid gradients that exist between various membrane compartments. Recent studies have highlighted the role of inositol phospholipids in the nonvesicular transport of lipids at membrane contact sites. This review attempts to summarize our current understanding of these complex lipid dynamics and highlights their implications for defining future research directions.

Roles of Phosphatidylinositol 4-Phosphorylation in Non-vesicular Cholesterol Trafficking.

Cholesterol (Chol) is an essential component of all eukaryotic cell membranes that affects the function of numerous peripheral as well as integral membrane proteins. Chol is synthesized in the ER, but it is selectively enriched within the plasma membrane (PM) and other endomembranes, which requires Chol to cross the aqueous phase of the cytoplasm. In addition to the classical vesicular trafficking pathways that are known to facilitate the bulk transport of membrane intermediates, Chol is also transported via non-vesicular lipid transfer proteins that work primarily within specialized membrane contact sites. Some of these transport pathways work against established concentration gradients and hence require energy. Recent studies highlight the unique role of phosphoinositides (PPIns), and phosphatidylinositol 4-phosphate (PI4P) in particular, for the control of non-vesicular Chol transport. In this chapter, we will review the emerging connection between Chol, PPIns, and lipid transfer proteins that include the important family of oxysterol-binding protein related proteins, or ORPs.

Myelination of peripheral nerves is controlled by PI4KB through regulation of Schwann cell Golgi function.

Better understanding myelination of peripheral nerves would benefit patients affected by peripheral neuropathies, including Charcot-Marie-Tooth disease. Little is known about the role the Golgi compartment plays in Schwann cell (SC) functions. Here, we studied the role of Golgi in myelination of peripheral nerves in mice through SC-specific genetic inactivation of phosphatidylinositol 4-kinase beta (PI4KB), a Golgi-associated lipid kinase. Sciatic nerves of such mice showed thinner myelin of large diameter axons and gross aberrations in myelin organization affecting the nodes of Ranvier, the Schmidt-Lanterman incisures, and Cajal bands. Nonmyelinating SCs showed a striking inability to engulf small diameter nerve fibers. SCs of mutant mice showed a distorted Golgi morphology and disappearance of OSBP at the cis-Golgi compartment, together with a complete loss of GOLPH3 from the entire Golgi. Accordingly, the cholesterol and sphingomyelin contents of sciatic nerves were greatly reduced and so was the number of caveolae observed in SCs. Although the conduction velocity of sciatic nerves of mutant mice showed an 80% decrease, the mice displayed only subtle impairment in their motor functions. Our analysis revealed that Golgi functions supported by PI4KB are critically important for proper myelination through control of lipid metabolism, protein glycosylation, and organization of microvilli in the nodes of Ranvier of peripheral nerves.

Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease progression in mice independent of blood pressure regulation.

Scavenger receptor CD36 participates in lipid metabolism and inflammatory pathways important for cardiovascular disease and chronic kidney disease (CKD). Few pharmacological agents are available to slow the progression of CKD. However, apolipoprotein A-I-mimetic peptide 5A antagonizes CD36 in vitro. To test the efficacy of 5A, and to test the role of CD36 during CKD, we compared wild-type to CD36 knockout mice and wild-type mice treated with 5A, in a progressive CKD model that resembles human disease. Knockout and 5A-treated wild-type mice were protected from CKD progression without changes in blood pressure and had reductions in cardiovascular risk surrogate markers that are associated with CKD. Treatment with 5A did not further protect CD36 knockout mice from CKD progression, implicating CD36 as its main site of action. In a separate model of kidney fibrosis, 5A-treated wild-type mice had less macrophage infiltration and interstitial fibrosis. Peptide 5A exerted anti-inflammatory effects in the kidney and decreased renal expression of inflammasome genes. Thus, CD36 is a new therapeutic target for CKD and its associated cardiovascular risk factors. Peptide 5A may be a promising new agent to slow CKD progression.

Molecular basis for plasma membrane recruitment of PI4KA by EFR3.

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIIIa/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease.

Combination therapy with an angiotensin II receptor blocker and an HMG-CoA reductase inhibitor in experimental subtotal nephrectomy.

BACKGROUND: Angiotensin receptor 1 blockers (ARB) are standard nephroprotective drugs in chronic kidney disease. There is less evidence for a nephroprotective effect of HMG-CoA reductase inhibitors (statins) and much less is known about potential benefits of combination therapy. We evaluated the therapeutic potential of a statin alone or in combination with an ARB in experimental chronic kidney disease. METHODS: Subtotally nephrectomized (5/6 Nx) rats were treated early with vehicle, losartan, cerivastatin or losartan/cerivastatin. Expression of messenger RNA (mRNA) was assessed by real-time reverse transcription-polymerase chain reaction. Tissue proteins were localized by immunohistochemistry. Nuclear factor-κB (NF-κB) activation was measured in whole kidneys. RESULTS: In contrast to the sham group, at 6 weeks, vehicle-treated 5/6 Nx rats displayed renal lesions, albuminuria and increased blood pressure, serum creatinine and total kidney NF-κB p65 DNA-binding activity and preproendothelin-1, fibronectin and type I and III collagen mRNA. NF-κB activation correlated with albuminuria and histological renal injury. Losartan or combination therapy preserved renal function, abrogated albuminuria and improved glomerular and interstitial histology. Cerivastatin alone preserved renal function and improved interstitial injury but did not influence albuminuria, glomerular histology or NF-κB activation. Losartan/cerivastatin normalized kidney NF-κB activation and extracellular matrix mRNA expression pattern. The effect of losartan alone on these parameters was less intense. All treatments decreased preproendothelin-1 mRNA and preserved interstitial capillaries. CONCLUSIONS: In a chronic kidney disease model, early treatment with either an ARB or a statin preserved renal function although the mechanisms differed. Combination therapy with an ARB and a statin did not confer clear-cut advantages on biochemical and histological parameters over ARB alone, although it further improved the kidney NF-κB and gene expression profile.

Schwann-Cell-Specific Deletion of Phosphatidylinositol 4-Kinase Alpha Causes Aberrant Myelination.

Active membrane remodeling during myelination relies on phospholipid synthesis and membrane polarization, both of which are known to depend on inositol phospholipids. Here, we show that sciatic nerves of mice lacking phosphatidylinositol 4-kinase alpha (PI4KA) in Schwann cells (SCs) show substantially reduced myelin thickness with grave consequences on nerve conductivity and motor functions. Surprisingly, prolonged inhibition of PI4KA in immortalized mouse SCs failed to decrease plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) levels or PI 3-kinase (PI3K) activation, in spite of large reductions in plasma membrane PI4P levels. Instead, it caused rearrangements of the actin cytoskeleton, which was also observed in sciatic nerves of knockout animals. PI4KA inactivation disproportionally reduced phosphatidylserine, phosphatidylethanolamine, and sphingomyelin content in mutant nerves, with similar changes observed in SCs treated with a PI4KA inhibitor. These studies define a role for PI4KA in myelin formation primarily affecting metabolism of key phospholipids and the actin cytoskeleton.

TLR4 mutant mice are protected from renal fibrosis and chronic kidney disease progression.

Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppression. In this study we tested the role of Toll-like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild-type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low-dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low-grade systemic inflammation, splenocyte apoptosis and increased expression of the immune inhibitory receptor PD-1 in the spleen, which were not observed in TLR4 mutant mice. In vitro, endotoxin (LPS) directly upregulated NLRP3 inflammasome expression in renal epithelial cells via TLR4. In summary, TLR4 contributes to renal fibrosis and CKD progression, at least in part, via inflammasome activation in renal epithelial cells, and may also participate in the dysregulated immune response that is associated with CKD.

Automated quantification of renal fibrosis with Sirius Red and polarization contrast microscopy.

Interstitial fibrosis is commonly measured by histology. The Masson trichrome stain is widely used, with semiquantitative scores subjectively assigned by trained operators. We have developed an objective technique combining Sirius Red staining, polarization contrast microscopy, and automated analysis. Repeated analysis of the same sections by the same operator (r = 0.99) or by different operators (r = 0.98) was highly consistent for Sirius Red, while Masson trichrome performed less consistently (r = 0.61 and 0.72, respectively). These techniques performed equally well when comparing sections from the left and right kidneys of mice. Poor correlation between Sirius Red and Masson trichrome may reflect different specificities, as enhanced birefringence with Sirius Red staining is specific for collagen type I and III fibrils. Combining whole-section imaging and automated image analysis with Sirius Red/polarization contrast is a rapid, reproducible, and precise technique that is complementary to Masson trichrome. It also prevents biased selection of fields as fibrosis is measured on the entire kidney section. This new tool shall enhance our search for novel therapeutics and noninvasive biomarkers for fibrosis.

Severe compensatory hyperhidrosis following thoracic sympathectomy successfully treated with low doses of botulinum toxin A.

Compensatory hyperhidrosis is an adverse effect of thoracic sympathectomy that can be debilitating, which is why an efficient treatment is demanded. Botulinum toxin is an emerging treatment, not well known yet. We report two cases of compensatory hyperhidrosis following thoracic sympathectomy which were both treated with low doses of botulinum toxin A. The patients, a male and a female, noted a high level of satisfaction with the abolishment of sweating that was maintained up to 10 months. We consider that low doses of botulinum toxin A is a well tolerated, safe and effective treatment for compensatory hyperhidrosis and should be offered as an alternative treatment.