Gesine Saher

High cholesterol level is essential for myelin membrane growth

Cholesterol in the mammalian brain is a risk factor for certain neurodegenerative diseases, raising the question of its normal function. In the mature brain, the highest cholesterol content is found in myelin. We therefore created mice that lack the ability to synthesize cholesterol in myelin-forming oligodendrocytes. Mutant oligodendrocytes survived, but CNS myelination was severely perturbed, and mutant mice showed ataxia and tremor. CNS myelination continued at a reduced rate for many months, and during this period, the cholesterol-deficient oligodendrocytes actively enriched cholesterol and assembled myelin with >70% of the cholesterol content of wild-type myelin. This shows that cholesterol is an indispensable component of myelin membranes and that cholesterol availability in oligodendrocytes is a rate-limiting factor for brain maturation.

Neuropsychiatric disease relevance of circulating anti-NMDA receptor autoantibodies depends on blood-brain barrier integrity.

In 2007, a multifaceted syndrome, associated with anti-NMDA receptor autoantibodies (NMDAR-AB) of immunoglobulin-G isotype, has been described, which variably consists of psychosis, epilepsy, cognitive decline and extrapyramidal symptoms. Prevalence and significance of NMDAR-AB in complex neuropsychiatric disease versus health, however, have remained unclear. We tested sera of 2817 subjects (1325 healthy, 1081 schizophrenic, 263 Parkinson and 148 affective-disorder subjects) for presence of NMDAR-AB, conducted a genome-wide genetic association study, comparing AB carriers versus non-carriers, and assessed their influenza AB status. For mechanistic insight and documentation of AB functionality, in vivo experiments involving mice with deficient blood–brain barrier (ApoE−/−) and in vitro endocytosis assays in primary cortical neurons were performed. In 10.5% of subjects, NMDAR-AB (NR1 subunit) of any immunoglobulin isotype were detected, with no difference in seroprevalence, titer or in vitro functionality between patients and healthy controls. Administration of extracted human serum to mice influenced basal and MK-801-induced activity in the open field only in ApoE−/− mice injected with NMDAR-AB-positive serum but not in respective controls. Seropositive schizophrenic patients with a history of neurotrauma or birth complications, indicating an at least temporarily compromised blood–brain barrier, had more neurological abnormalities than seronegative patients with comparable history. A common genetic variant (rs524991, P=6.15E−08) as well as past influenza A (P=0.024) or B (P=0.006) infection were identified as predisposing factors for NMDAR-AB seropositivity. The >10% overall seroprevalence of NMDAR-AB of both healthy individuals and patients is unexpectedly high. Clinical significance, however, apparently depends on association with past or present perturbations of blood–brain barrier function.

The PreS2 activator MHBst of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice

The large hepatitis B virus (HBV) surface protein (LHBs) and C‐terminally truncated middle size surface proteins (MHBst) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBst activators are paradigmatic for this class of activators. Here we report that MHBst is protein kinase C (PKC)‐dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBst triggers PKC‐dependent activation of c‐Raf‐1/Erk2 signaling that is a prerequisite for MHBst‐dependent activation of AP‐1 and NF‐κB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBst specifically in the liver. In these mice, a permanent PreS2‐dependent specific activation of c‐Raf‐1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBst exert a tumor promoter‐like function by activation of key enzymes of proliferation control.

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

ABSTRACT Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Lipid metabolism in myelinating glial cells: lessons from human inherited disorders and mouse models

The integrity of central and peripheral nervous system myelin is affected in numerous lipid metabolism disorders. This vulnerability was so far mostly attributed to the extraordinarily high level of lipid synthesis that is required for the formation of myelin, and to the relative autonomy in lipid synthesis of myelinating glial cells because of blood barriers shielding the nervous system from circulating lipids. Recent insights from analysis of inherited lipid disorders, especially those with prevailing lipid depletion and from mouse models with glia-specific disruption of lipid metabolism, shed new light on this issue. The particular lipid composition of myelin, the transport of lipid-associated myelin proteins, and the necessity for timely assembly of the myelin sheath all contribute to the observed vulnerability of myelin to perturbed lipid metabolism. Furthermore, the uptake of external lipids may also play a role in the formation of myelin membranes. In addition to an improved understanding of basic myelin biology, these data provide a foundation for future therapeutic interventions aiming at preserving glial cell integrity in metabolic disorders.

Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction

Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.

Secondary reduction of α7B integrin in laminin α2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The α7β1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the α7 integrin are laminin-1 (α1-β1-γ1), laminin-2 (α2-β1-γ1) and laminin-4 (α2-β2-γ1). Targeted deletion of the gene for the α7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the α7B and β1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the α7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the β1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of α7B integrin was significantly reduced at the sarcolemma in six patients with laminin α2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin α2 chain expressed. In contrast, the expression of the laminin α2 chain was not altered in the skeletal muscle of the α7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the α7 integrin subunit and that of the laminin α2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of α7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the β1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of β1D integrin was significantly reduced in the α7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of α7B in laminin α2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in α7 deficient mice, implies that the α7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Survival of adult neurons lacking cholesterol synthesis in vivo

BackgroundCholesterol, an essential component of all mammalian plasma membranes, is highly enriched in the brain. Both during development and in the adult, brain cholesterol is derived from local cholesterol synthesis and not taken up from the circulation. However, the contribution of neurons and glial cells to total brain cholesterol metabolism is unknown.ResultsUsing conditional gene inactivation in the mouse, we disrupted the squalene synthase gene (fdft1), which is critical for cholesterol synthesis, in cerebellar granule cells and some precerebellar nuclei. Mutant mice showed no histological signs of neuronal degeneration, displayed ultrastructurally normal synapses, and exhibited normal motor coordination. This revealed that these adult neurons do not require cell-autonomous cholesterol synthesis for survival or function.ConclusionWe conclude that at least some adult neurons no longer require endogenous cholesterol synthesis and can fully meet their cholesterol needs by uptake from their surrounding. Glia are a likely source of cholesterol in the central nervous system.

A critical role for the cholesterol-associated proteolipids PLP and M6B in myelination of the central nervous system

The formation of central nervous system myelin by oligodendrocytes requires sterol synthesis and is associated with a significant enrichment of cholesterol in the myelin membrane. However, it is unknown how oligodendrocytes concentrate cholesterol above the level found in nonmyelin membranes. Here, we demonstrate a critical role for proteolipids in cholesterol accumulation. Mice lacking the most abundant myelin protein, proteolipid protein (PLP), are fully myelinated, but PLP‐deficient myelin exhibits a reduced cholesterol content. We therefore hypothesized that “high cholesterol” is not essential in the myelin sheath itself but is required for an earlier step of myelin biogenesis that is fully compensated for in the absence of PLP. We also found that a PLP‐homolog, glycoprotein M6B, is a myelin component of low abundance. By targeting the Gpm6b‐gene and crossbreeding, we found that single‐mutant mice lacking either PLP or M6B are fully myelinated, while double mutants remain severely hypomyelinated, with enhanced neurodegeneration and premature death. As both PLP and M6B bind membrane cholesterol and associate with the same cholesterol‐rich oligodendroglial membrane microdomains, we suggest a model in which proteolipids facilitate myelination by sequestering cholesterol. While either proteolipid can maintain a threshold level of cholesterol in the secretory pathway that allows myelin biogenesis, lack of both proteolipids results in a severe molecular imbalance of prospective myelin membrane. However, M6B is not efficiently sorted into mature myelin, in which it is 200‐fold less abundant than PLP. Thus, only PLP contributes to the high cholesterol content of myelin by association and co‐transport.

Therapy of Pelizaeus-Merzbacher disease in mice by feeding a cholesterol-enriched diet

Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.