Another Use for a Proven Drug: Experimental Evidence for the Potential of Artemisinin and Its Derivatives to Treat Alzheimer's Disease.

Plant-derived multitarget compounds may represent a promising therapeutic strategy for multifactorial diseases, such as Alzheimer's disease (AD). Artemisinin and its derivatives were indicated to beneficially modulate various aspects of AD pathology in different AD animal models through the regulation of a wide range of different cellular processes, such as energy homeostasis, apoptosis, proliferation and inflammatory pathways. In this review, we aimed to provide an up-to-date overview of the experimental evidence documenting the neuroprotective activities of artemi-sinins to underscore the potential of these already-approved drugs for treating AD also in humans and propose their consideration for carefully designed clinical trials. In particular, the benefits to the main pathological hallmarks and events in the pathological cascade throughout AD development in different animal models of AD are summarized. Moreover, dose- and context-dependent effects of artemisinins are noted.

Primary cilia are critical for tracheoesophageal septation.

INTRODUCTION: Primary cilia play pivotal roles in the patterning and morphogenesis of a wide variety of organs during mammalian development. Here we examined murine foregut septation in the cobblestone mutant, a hypomorphic allele of the gene encoding the intraflagellar transport protein IFT88, a protein essential for normal cilia function. RESULTS: We reveal a crucial role for primary cilia in foregut division, since their dramatic decrease in cilia in both the foregut endoderm and mesenchyme of mutant embryos resulted in a proximal tracheoesophageal septation defects and in the formation of distal tracheo(broncho)esophageal fistulae similar to the most common congenital tracheoesophageal malformations in humans. Interestingly, the dorsoventral patterning determining the dorsal digestive and the ventral respiratory endoderm remained intact, whereas Hedgehog signaling was aberrantly activated. CONCLUSIONS: Our results demonstrate the cobblestone mutant to represent one of the very few mouse models that display both correct endodermal dorsoventral specification but defective compartmentalization of the proximal foregut. It stands exemplary for a tracheoesophageal ciliopathy, offering the possibility to elucidate the molecular mechanisms how primary cilia orchestrate the septation process. The plethora of malformations observed in the cobblestone embryo allow for a deeper insight into a putative link between primary cilia and human VATER/VACTERL syndromes.

Loss of Extrasynaptic Inhibitory Glycine Receptors in the Hippocampus of an AD Mouse Model Is Restored by Treatment with Artesunate.

Alzheimer's disease (AD) is characterized by synaptic failure and neuronal loss. Recently, we demonstrated that artemisinins restored the levels of key proteins of inhibitory GABAergic synapses in the hippocampus of APP/PS1 mice, a model of cerebral amyloidosis. In the present study, we analyzed the protein levels and subcellular localization of α2 and α3 subunits of GlyRs, indicated as the most abundant receptor subtypes in the mature hippocampus, in early and late stages of AD pathogenesis, and upon treatment with two different doses of artesunate (ARS). Immunofluorescence microscopy and Western blot analysis demonstrated that the protein levels of both α2 and α3 GlyRs are considerably reduced in the CA1 and the dentate gyrus of 12-month-old APP/PS1 mice when compared to WT mice. Notably, treatment with low-dose ARS affected GlyR expression in a subunit-specific way; the protein levels of α3 GlyR subunits were rescued to about WT levels, whereas that of α2 GlyRs were not affected significantly. Moreover, double labeling with a presynaptic marker indicated that the changes in GlyR α3 expression levels primarily involve extracellular GlyRs. Correspondingly, low concentrations of artesunate (≤1 µM) also increased the extrasynaptic GlyR cluster density in hAPPswe-transfected primary hippocampal neurons, whereas the number of GlyR clusters overlapping presynaptic VIAAT immunoreactivities remained unchanged. Thus, here we provide evidence that the protein levels and subcellular localization of α2 and α3 subunits of GlyRs show regional and temporal alterations in the hippocampus of APP/PS1 mice that can be modulated by the application of artesunate.

Artemisinin-treatment in pre-symptomatic APP-PS1 mice increases gephyrin phosphorylation at Ser270: a modification regulating postsynaptic GABAAR density.

Artemisinins, a group of plant-derived sesquiterpene lactones, are efficient antimalarial agents. They also share anti-inflammatory and anti-viral activities and were considered for treatment of neurodegenerative disorders like Alzheimer's disease (AD). Additionally, artemisinins bind to gephyrin, the multifunctional scaffold of GABAergic synapses, and modulate inhibitory neurotransmission in vitro. We previously reported an increased expression of gephyrin and GABAA receptors in early pre-symptomatic stages of an AD mouse model (APP-PS1) and in parallel enhanced CDK5-dependent phosphorylation of gephyrin at S270. Here, we studied the effects of artemisinin on gephyrin in the brain of young APP-PS1 mice. We detected an additional increase of gephyrin protein level, elevated gephyrin phosphorylation at Ser270, and an increased amount of GABAAR-γ2 subunits after artemisinin-treatment. Interestingly, the CDK5 activator p35 was also upregulated. Moreover, we demonstrate decreased density of postsynaptic gephyrin and GABAAR-γ2 immunoreactivities in cultured hippocampal neurons expressing gephyrin with alanine mutations at two CDK5 phosphorylation sites. In addition, the activity-dependent modulation of synaptic protein density was abolished in neurons expressing gephyrin lacking one or both of these phosphorylation sites. Thus, our results reveal that artemisinin modulates expression as well as phosphorylation of gephyrin at sites that might have important impact on GABAergic synapses in AD.

Artesunate restores the levels of inhibitory synapse proteins and reduces amyloid-ß and C-terminal fragments (CTFs) of the amyloid precursor protein in an AD-mouse model.

Alzheimer's disease (AD) is the most frequent form of dementia, characterized histopathologically by the formation of amyloid plaques and neurofibrillary tangles in the brain. Amyloid ß-peptide (Aß) is a major component of amyloid plaques and is released together with carboxy-terminal fragments (CTFs) from the amyloid precursor protein (APP) through proteolytic cleavage, thought to contribute to synapse dysfunction and loss along the progression of AD. Artemisinins, primarily antimalarial drugs, reduce neuroinflammation and improve cognitive capabilities in mouse models of AD. Furthermore, artemisinins were demonstrated to target gephyrin, the main scaffold protein of inhibitory synapses and modulate GABAergic neurotransmission in vitro. Previously, we reported a robust decrease of inhibitory synapse proteins in the hippocampus of 12-month-old double transgenic APP-PS1 mice which overexpress in addition to the Swedish mutated form of the human APP a mutated presenilin 1 (PS1) gene and are characterized by a high plaque load at this age. Here, we provide in vivo evidence that treating these mice with artemisinin or its semisynthetic derivative artesunate in two different doses (10 mg/kg and 100 mg/kg), these compounds affect differently inhibitory synapse components, amyloid plaque load and APP-processing. Immunofluorescence microscopy demonstrated the rescue of gephyrin and γ2-GABAA-receptor protein levels in the brain of treated mice with both, artemisinin and artesunate, most efficiently with a low dose of artesunate. Remarkably, artemisinin reduced only in low dose the amyloid plaque load correlating with lower levels of mutated human APP (hAPPswe) whereas artesunate treatment in both doses resulted in significantly lower plaque numbers. Correspondingly, the level of APP-cleavage products, specifically the amount of CTFs in hippocampus homogenates was reduced significantly only by artesunate, in line with the findings in hAPPswe expressing cultured hippocampal neurons evidencing a concentration-dependent inhibition of CTF-release by artesunate already in the nanomolar range. Thus, our data support artemisinins as neuroprotective multi-target drugs, exhibiting a potent anti-amyloidogenic activity and reinforcing key proteins of inhibitory synapses.

Amyloid-ß Fosters p35/CDK5 Signaling Contributing to Changes of Inhibitory Synapses in Early Stages of Cerebral Amyloidosis.

Early changes in inhibitory synapse connectivities are thought to contribute to the excitation/inhibition imbalance preceding neurodegeneration in Alzheimer's disease (AD). Recently, we reported a robust increase in the level of different key-proteins of inhibitory synapses in hippocampal subregions of pre-symptomatic APPswe-PS1 mice, a model of cerebral amyloidosis. Besides increased inhibitory synaptic clusters on parvalbumin-positive projections in CA1 and CA3, we observed impaired communication between these two hippocampal areas of young APP-PS1 mice. Interestingly, the phosphorylation of gephyrin, a major organizer of inhibitory synapses, was also increased. Here, we demonstrate that the protein levels of CDK5, a kinase involved in the phosphorylation of gephyrin, and its regulatory protein p35 are also significantly increased in hippocampal subregions of young APP-PS1 mice. Consistently, the expression of hAPP-swe in cultured hippocampal neurons resulted in higher p35-protein levels, indicating a possible molecular link between increased Aß-production and the elevated p35/CDK5 levels seen in vivo. Further, a shRNA mediated downregulation of p35-expression in hippocampal neurons correlated with a decrease in gephyrin phosphorylation and in a reduced density of synaptic γ2-GABAA-receptor clusters. These findings, together with the detection of gephyrin colocalization with CDK5 and p35 by immunostaining and proximity ligation experiments in vivo and in vitro, are supporting our hypothesis that Aß has a profound impact on inhibitory network properties, likely mediated at least in part by p35/CDK5 signaling. This further underscores the impact of altered inhibitory synaptic transmission in AD.

Early alterations in hippocampal perisomatic GABAergic synapses and network oscillations in a mouse model of Alzheimer's disease amyloidosis.

Several lines of evidence imply changes in inhibitory interneuron connectivity and subsequent alterations in oscillatory network activities in the pathogenesis of Alzheimer's Disease (AD). Recently, we provided evidence for an increased immunoreactivity of both the postsynaptic scaffold protein gephyrin and the GABAA receptor γ2-subunit in the hippocampus of young (1 and 3 months of age), APPPS1 mice. These mice represent a well-established model of cerebral amyloidosis, which is a hallmark of human AD. In this study, we demonstrate a robust increase of parvalbumin immunoreactivity and accentuated projections of parvalbumin positive (PV+) interneurons, which target perisomatic regions of pyramidal cells within the hippocampal subregions CA1 and CA3 of 3-month-old APPPS1 mice. Colocalisation studies confirmed a significant increase in the density of PV+ projections labeled with antibodies against a presynaptic (vesicular GABA transporter) and a postsynaptic marker (gephyrin) of inhibitory synapses within the pyramidal cell layer of CA1 and CA3. As perisomatic inhibition by PV+-interneurons is crucial for the generation of hippocampal network oscillations involved in spatial processing, learning and memory formation we investigated the impact of the putative enhanced perisomatic inhibition on two types of fast neuronal network oscillations in acute hippocampal slices: 1. spontaneously occurring sharp wave-ripple complexes (SPW-R), and 2. cholinergic γ-oscillations. Interestingly, both network patterns were generally preserved in APPPS1 mice similar to WT mice. However, the comparison of simultaneous CA3 and CA1 recordings revealed that the incidence and amplitude of SPW-Rs were significantly lower in CA1 vs CA3 in APPPS1 slices, whereas the power of γ-oscillations was significantly higher in CA3 vs CA1 in WT-slices indicating an impaired communication between the CA3 and CA1 network activities in APPPS1 mice. Taken together, our data demonstrate an increased GABAergic synaptic output of PV+ interneurons impinging on pyramidal cells of CA1 and CA3, which might limit the coordinated cross-talk between these two hippocampal areas in young APPPS1 mice and mediate long-term changes in synaptic inhibition during progression of amyloidosis.

Biphasic Alteration of the Inhibitory Synapse Scaffold Protein Gephyrin in Early and Late Stages of an Alzheimer Disease Model.

The pathogenesis of Alzheimer disease (AD) is thought to begin many years before the diagnosis of dementia. Accumulating evidence indicates the involvement of GABAergic neurotransmission in the physiopathology of AD. However, in comparison to excitatory synapses, the structural and functional alterations of inhibitory synapses in AD are less well characterized. We studied the expression and distribution of proteins specific for inhibitory synapses in hippocampal areas of APPPS1 mice at different ages. Interestingly, by immunoblotting and confocal fluorescence microscopy, we disclosed a robust increase in the expression of gephyrin, an organizer of ligand-gated ion channels at inhibitory synapses in hippocampus CA1 and dentate gyrus of young presymptomatic APPPS1 mice (1 to 3 months) as compared to controls. The postsynaptic γ2-GABA(A)-receptor subunit and the presynaptic vesicular inhibitory amino acid transporter protein showed similar expression patterns. In contrast, adult transgenic animals (12 months) displayed decreased levels of these proteins in comparison to wild type in hippocampus areas devoid of amyloid plaques. Within most plaques, strong gephyrin immunoreactivity was detected, partially colocalizing with vesicular amino acid transporter and GABA(A)-receptor γ2 subunit immunoreactivities. Our results indicate a biphasic alteration in expression of hippocampal inhibitory synapse components in AD. Altered inhibition of neurotransmission might be an early prognostic marker and might even be involved in the pathogenesis of AD.

Male meiotic cytokinesis requires ceramide synthase 3-dependent sphingolipids with unique membrane anchors.

Somatic cell cytokinesis was shown to involve the insertion of sphingolipids (SLs) to midbodies prior to abscission. Spermatogenic midbodies transform into stable intercellular bridges (ICBs) connecting clonal daughter cells in a syncytium. This process requires specialized SL structures. (1) Using high resolution-mass spectrometric imaging, we show in situ a biphasic pattern of SL synthesis with testis-specific anchors. This pattern correlates with and depends on ceramide synthase 3 (CerS3) localization in both, pachytene spermatocytes until completion of meiosis and elongating spermatids. (2) Blocking the pathways to germ cell-specific ceramides (CerS3-KO) and further to glycosphingolipids (glucosylceramide synthase-KO) in mice highlights the need for special SLs for spermatid ICB stability. In contrast to somatic mitosis these SLs require ultra-long polyunsaturated anchors with unique physico-chemical properties, which can only be provided by CerS3. Loss of these anchors causes enhanced apoptosis during meiosis, formation of multinuclear giant cells and spermatogenic arrest. Hence, testis-specific SLs, which we also link to CerS3 in human testis, are quintessential for male fertility.

Ceramide synthesis in the epidermis.

The epidermis and in particular its outermost layer the stratum corneum provides terrestrial vertebrates with a pivotal defensive barrier against water loss, xenobiotics and harmful pathogens. A vital demand for this epidermal permeability barrier is the lipid-enriched lamellar matrix that embeds the enucleated corneocytes. Ceramides are the major components of these highly ordered intercellular lamellar structures, in which linoleic acid- and protein-esterified ceramides are crucial for structuring and maintaining skin barrier integrity. In this review, we describe the fascinating diversity of epidermal ceramides including 1-O-acylceramides. We focus on epidermal ceramide biosynthesis emphasizing its metabolic and topological requirements and discuss enzymes that may be involved in α- and ω-hydroxylation. Finally, we turn to epidermal ceramide regulation, highlighting transcription factors and liposensors recently described to play crucial roles in modulating skin lipid metabolism and epidermal barrier homeostasis. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier.

Multiple essential roles for primary cilia in heart development.

BACKGROUND: The primary cilium is a microtubule-based, plasma membrane-ensheathed protrusion projecting from the basal bodies of almost all cell types in the mammalian body. In the past several years a plethora of papers has indicated a crucial role for primary cilia in the development of a wide variety of organs. We have investigated heart development in cobblestone, a hypomorphic allele of the gene encoding the intraflagellar transport protein Ift88, and uncovered a number of the most common congenital heart defects seen in newborn humans. METHODS: We generated serial sections of mutant cobblestone and wild type embryos in the region encompassing the heart and the cardiac outflow tract. The sections were further processed to generate three-dimensional reconstructions of these structures, and immunofluorescence confocal microscopy, transmission electron microscopy, and in situ hybridization were used to examine signal transduction pathways in the relevant areas. Whole mount in situ hybridization was also employed for certain developmental markers. RESULTS: In addition to an enlarged pericardium and failure of both ventricular and atrial septum formation, the cobblestone mutants displayed manifold defects in outflow tract formation, including persistent truncus arteriosus, an overriding aorta, and abnormal transformation of the aortic arches. To discern the basis of these anomalies we examined both the maintenance of primary cilia as well as endogenous and migratory embryonic cell populations that contribute to the outflow tract and atrioventricular septa. The colonization of the embryonic heart by cardiac neural crest occurred normally in the cobblestone mutant, as did the expression of Sonic hedgehog. However, with the loss of primary cilia in the mutant hearts, there was a loss of both downstream Sonic hedgehog signaling and of Islet 1 expression in the second heart field, a derivative of the pharyngeal mesoderm. In addition, defects were recorded in development of atrial laterality and ventricular myocardiogenesis. Finally, we observed a reduction in expression of Bmp4 in the outflow tract, and complete loss of expression of both Bmp2 and Bmp4 in the atrioventricular endocardial cushions. Loss of BMP2/4 signaling may result in the observed proliferative defect in the endocardial cushions, which give rise to both the atrioventricular septa as well as to the septation of the outflow tract. CONCLUSIONS: Taken together, our results potentially identify a novel link between Sonic hedgehog signaling at the primary cilium and BMP-dependent effects upon cardiogenesis. Our data further point to a potential linkage of atrioventricular septal defects, the most common congenital heart defects, to genes of the transport machinery or basal body of the cilia.

Loss of ceramide synthase 3 causes lethal skin barrier disruption.

The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1-6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton.

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice.

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell's intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment.

Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse cerebellum.

Ether lipids (ELs), particularly plasmalogens, are essential constituents of the mammalian central nervous system. The physiological role of ELs, in vivo, however is still enigmatic. In the present study, we characterized a mouse model carrying a targeted deletion of the peroxisomal dihydroxyacetonephosphate acyltransferase gene that results in the complete lack of ELs. Investigating the cerebellum of these mice, we observed: (i) defects in foliation patterning and delay in precursor granule cell migration, (ii) defects in myelination and concomitant reduction in the level of myelin basic protein, (iii) disturbances in paranode organization by extending the Caspr distribution and disrupting axo-glial septate-like junctions, (iv) impaired innervation of Purkinje cells by both parallel fibers and climbing fibers and (v) formation of axon swellings by the accumulation of inositol-tris-phosphate receptor 1 containing smooth ER-like tubuli. Functionally, conduction velocity of myelinated axons in the corpus callosum was significantly reduced. Most of these phenotypes were already apparent at P20 but still persisted in 1-year-old animals. In summary, these data show that EL deficiency results in severe developmental and lasting structural alterations at the cellular and network level of the cerebellum, and reveal an important role of ELs for proper brain function. Common molecular mechanisms that may underlie these phenotypes are discussed.

A crucial role for primary cilia in cortical morphogenesis.

Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70-80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain.

Male germ cells require polyenoic sphingolipids with complex glycosylation for completion of meiosis: a link to ceramide synthase-3.

Previously, it was found that a novel class of neutral fucosylated glycosphingolipids (GSLs) is required for male fertility. These lipids contain very long-chain (C26-C32) polyunsaturated (4-6 double bonds) fatty acid residues (VLC-PUFAs). To assess the role of these complex GSLs in spermatogenesis, we have now investigated with which of the testicular cell types these lipids are associated. During postnatal development, complex glycosylated and simple VLC-PUFA sphingolipids were first detectable at day 15, when the most advanced germ cells are pachytene spermatocytes. Their synthesis is most likely driven by ceramide synthase-3. This enzyme is encoded by the Cers3/Lass3 gene (longevity assurance genes), and out of six members of this gene family, only Cers3 mRNA expression was limited to germ cells, where it was up-regulated more than 700-fold during postnatal testicular maturation. Increasing levels of neutral complex VLC-PUFA GSLs also correlated with the progression of spermatogenesis in a series of male sterile mutants with arrests at different stages of spermatogenesis. Remarkably, fucosylation of the complex VLC-PUFA GSLs was not essential for spermatogenesis, as fucosylation-deficient mice produced nonfucosylated versions of the complex testicular VLC-PUFA GSLs, had complete spermatogenesis, and were fertile. Nevertheless, sterile Galgt1(-/-) mice, with a defective meiotic cytokinesis and a subsequent block in spermiogenesis, lacked complex but contained simple VLC-PUFA GSLs, as well as VLC-PUFA ceramides and sphingomyelins, indicating that the latter lipids are not sufficient for completion of spermatogenesis. Thus, our data imply that both glycans and the particular acyl chains of germinal sphingolipids are relevant for proper completion of meiosis.

Effects of laparoscopic division of spermatic vessels on histological changes of testes: long-term observation in the model of prepubertal rat.

Laparoscopic Fowler-Stephens and Palomo procedures are now commonly performed in children with high positioned intra-abdominal cryptorchidism and varicocele, respectively. During the procedures, the spermatic vessels are ligated and therefore the question of risk related to testicular atrophy is often raised. The long-term follow-up of the histology after the procedures is rare. In this study, we simulated a laparoscopic spermatic vessels clipping and division (SVCD) in a prepubertal rat model, and examined the histological alterations of the testes with regard to spermatogenic arrest between prepuberty and middle age. Thirty-day-old Wistar rats divided randomly into three groups underwent laparoscopic sham operation, unilateral SVCD and unilateral SVCD with additional contralateral orchiectomy, respectively. Histological investigations observed on semithin and paraffin sections were performed at seven different postoperative intervals between day 9 and day 540. We defined partial, most and complete spermatogenic arrest of the seminiferous tubules to correspond with mild, severe spermatogenic arrest and atrophy, respectively. Laparoscopic SVCD induced testicular spermatogenic arrest in a total of 85% of the operated testes with different severity; 27% of operated testes with mild or severe spermatogenic arrest were seen between puberty and middle age (day 45-540 postoperative), and their size was only slightly reduced. Of the operated testes, 51% showed atrophic signs with a striking decrease in size, and their contralateral testes revealed in all cases mild or severe spermatogenic arrest started as early as day 45 postoperatively. Parallel to the spermatogenic arrest, Leydig cell hyperplasia developed frequently in impaired testes, especially in those without contralateral testes, finally reaching a typical adenoma size. Laparoscopic SVCD in prepubertal rats could disturb spermatogenesis with differing severity in most cases. This impairment could persist from peripuberty to middle age, and even involve the contralateral testes, in the case of operated testes and show complete spermatogenic arrest. This study showed that laparoscopic SVCD may have high risk in compromising the operated testis.

The ether lipid-deficient mouse: tracking down plasmalogen functions.

Chemical and physico-chemical properties as well as physiological functions of major mammalian ether-linked glycerolipids, including plasmalogens were reviewed. Their chemical structures were described and their effect on membrane fluidity and membrane fusion discussed. The recent generation of mouse models with ether lipid deficiency offered the possibility to study ether lipid and particularly plasmalogen functions in vivo. Ether lipid-deficient mice revealed severe phenotypic alterations, including arrest of spermatogenesis, development of cataract and defects in central nervous system myelination. In several cell culture systems lack of plasmalogens impaired intracellular cholesterol distribution affecting plasma membrane functions and structural changes of ER and Golgi cisternae. Based on these phenotypic anomalies that were accurately described conclusions were drawn on putative functions of plasmalogens. These functions were related to cell-cell or cell-extracellular matrix interactions, formation of lipid raft microdomains and intracellular cholesterol homeostasis. There are several human disorders, such as Zellweger syndrome, rhizomelic chondrodysplasia punctata, Alzheimer's disease, Down syndrome, and Niemann-Pick type C disease that are distinguished by altered tissue plasmalogen concentrations. The role plasmalogens might play in the pathology of these disorders is discussed.

Peroxisome biogenesis: where Arf and coatomer might be involved.

The present review summarizes recent observations on binding of Arf and COPI coat to isolated rat liver peroxisomes. The general structural and functional features of both Arf and coatomer were considered along with the requirements and dependencies of peroxisomal Arf and coatomer recruitment. Studies on the expression of mammalian Pex11 proteins, mainly Pex11alpha and Pex11beta, intimately related to the process of peroxisome proliferation, revealed a sequence of individual steps including organelle elongation/tubulation, formation of membrane and matrix protein patches segregating distinct proteins from each other, development of membrane constrictions and final membrane fission. Based on the similarities of the processes leading to cargo selection and concentration on Golgi membranes on the one hand and to the formation of peroxisomal protein patches on the other hand, an implication of Arf and COPI in distinct processes of peroxisomal proliferation is hypothesized. Alternatively, peroxisomal Arf/COPI might facilitate the formation of COPI-coated peroxisomal vesicles functioning in cargo transport and retrieval from peroxisomes to the ER. Recent observations suggesting transport of Pex3 and Pex19 during early steps of peroxisome biogenesis from the ER to peroxisomes inevitably propose such a retrieval mechanism, provided the ER to peroxisome pathway is based on transporting vesicles.