Peter Heimann

Mutation of Vps54 causes motor neuron disease and defective spermiogenesis in the wobbler mouse

Vacuolar-vesicular protein sorting (Vps) factors are involved in vesicular trafficking in eukaryotic cells. We identified the missense mutation L967Q in Vps54 in the wobbler mouse, an animal model of amyotrophic lateral sclerosis, and also characterized a lethal allele, Vps54β-geo. Motoneuron survival and spermiogenesis are severely compromised in the wobbler mouse, indicating that Vps54 has an essential role in these processes.

Isolation of Novel Multipotent Neural Crest-Derived Stem Cells from Adult Human Inferior Turbinate

Adult human neural crest-derived stem cells (NCSCs) are of extraordinary high plasticity and promising candidates for the use in regenerative medicine. Here we describe for the first time a novel neural crest-derived stem cell population within the respiratory epithelium of human adult inferior turbinate. In contrast to superior and middle turbinates, high amounts of source material could be isolated from human inferior turbinates. Using minimally-invasive surgery methods isolation is efficient even in older patients. Within their endogenous niche, inferior turbinate stem cells (ITSCs) expressed high levels of nestin, p75(NTR), and S100. Immunoelectron microscopy using anti-p75 antibodies displayed that ITSCs are of glial origin and closely related to nonmyelinating Schwann cells. Cultivated ITSCs were positive for nestin and S100 and the neural crest markers Slug and SOX10. Whole genome microarray analysis showed pronounced differences to human ES cells in respect to pluripotency markers OCT4, SOX2, LIN28, and NANOG, whereas expression of WDR5, KLF4, and c-MYC was nearly similar. ITSCs were able to differentiate into cells with neuro-ectodermal and mesodermal phenotype. Additionally ITSCs are able to survive and perform neural crest typical chain migration in vivo when transplanted into chicken embryos. However ITSCs do not form teratomas in severe combined immunodeficient mice. Finally, we developed a separation strategy based on magnetic cell sorting of p75(NTR) positive ITSCs that formed larger neurospheres and proliferated faster than p75(NTR) negative ITSCs. Taken together our study describes a novel, readily accessible source of multipotent human NCSCs for potential cell-replacement therapy.

Fine structure and molting of aesthetasc sense organs on the antennules of the isopod, Asellus aquaticus (Crustacea).

SummaryIn Asellus aquaticus certain distal antennular segments bear single sensilla referred to as aesthetascs. These show a proximal stem and a distal bulbous region. Depending on its position, each aesthetasc is innervated by either 50–60 or 70–80 bipolar sensory cells, the perikarya of which are situated within the pedunculus. Within the antennular segment the dendrites develop unbranched cilia (9 × 2 + 0 structure). The sensory cells are unusual in that mono- as well as biciliary dendrites are present within a single aesthetasc, the ratio of both types being correlated with the number of sensory cells. Cilia and receptor lymph cavity are enveloped by a set of 3–4 inner and 13–14 outer sheath cells, which terminate at the base of the sensillum, so that the delicate and poreless cuticle of the bulbous region encloses only outer segments within the receptor lymph fluid.A new molting type in arthropods is described in which the outer sheath cells alone build the new cuticle, whereas the inner sheath cells most probably have a protective function.A definition of aesthetascs is proposed based on finestructural criteria. Functionally the sensilla are considered to be chemoreceptors. This assumption is confirmed by experiments with diluted vital dye as well as lanthanum showing that dissolved substances penetrate the poreless cuticle instantaneously.

Schwann Cells Can Be Reprogrammed to Multipotency by Culture

Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions.

Regrowing the Adult Brain: NF-κB Controls Functional Circuit Formation and Tissue Homeostasis in the Dentate Gyrus

Cognitive decline during aging is correlated with a continuous loss of cells within the brain and especially within the hippocampus, which could be regenerated by adult neurogenesis. Here we show that genetic ablation of NF-κB resulted in severe defects in the neurogenic region (dentate gyrus) of the hippocampus. Despite increased stem cell proliferation, axogenesis, synaptogenesis and neuroprotection were hampered, leading to disruption of the mossy fiber pathway and to atrophy of the dentate gyrus during aging. Here, NF-κB controls the transcription of FOXO1 and PKA, regulating axogenesis. Structural defects culminated in behavioral impairments in pattern separation. Re-activation of NF-κB resulted in integration of newborn neurons, finally to regeneration of the dentate gyrus, accompanied by a complete recovery of structural and behavioral defects. These data identify NF-κB as a crucial regulator of dentate gyrus tissue homeostasis suggesting NF-κB to be a therapeutic target for treating cognitive and mood disorders.

Growth and differentiation of permanent and secondary mouse myogenic cell lines on microcarriers.

Abstract. Myogenesis involves the determination of progenitor cells to myoblasts, their fusion to yield multinuclear myotubes, and the maturation of myotubes to muscle fibres. This development is reflected in a time pattern of gene expression, e.g. of genes coding for desmin, the myogenic factors myogenin and myoD, the acetylcholine receptor α-subunit and the muscular chloride channel ClC-1. We attempted to improve yields and myogenic differentiation in culture by using three-dimensional microcarrier systems. Out of a variety of carriers tested in stationary cultures, collagen-coated dextran Cytodex3 beads proved optimal for the proliferation and differentiation of the murine myogenic cell line C2C12. With C2C12 myoblasts in stationary and stirred systems (Spinner- and SuperSpinner flasks), surface adherence, differentiation into myotubes and expression of muscle-specific mRNAs on Cytodex3 beads were the same as in conventional cultures. Other carriers tested (DEAE cellulose, glass, plastic, cellulose, polyester) did not support growth and differentiation of C2C12 cells. The secondary mouse myogenic stem cells M12 and M2.7-MDX proliferated and differentiated well in stationary Cytodex3 cultures, but no differentiation occurred in Spinner flasks. As indicated by light and scanning electron microscopy, C2C12 myotubes formed not only on but also in between Cytodex beads. The secondary cell lines may succumb to shear forces under these conditions.

Endosomal accumulation of APP in wobbler motor neurons reflects impaired vesicle trafficking: Implications for human motor neuron disease

BackgroundThe cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown but hypotheses about disease mechanisms include oxidative stress, defective axonal transport, mitochondrial dysfunction and disrupted RNA processing. Whereas familial ALS is well represented by transgenic mutant SOD1 mouse models, the mouse mutant wobbler (WR) develops progressive motor neuron degeneration due to a point mutation in the Vps54 gene, and provides an animal model for sporadic ALS. VPS54 protein as a component of a protein complex is involved in vesicular Golgi trafficking; impaired vesicle trafficking might also be mechanistic in the pathogenesis of human ALS.ResultsIn motor neurons of homozygous symptomatic WR mice, a massive number of endosomal vesicles significantly enlarged (up to 3 μm in diameter) were subjected to ultrastructural analysis and immunohistochemistry for the endosome-specific small GTPase protein Rab7 and for amyloid precursor protein (APP). Enlarged vesicles were neither detected in heterozygous WR nor in transgenic SOD1(G93A) mice; in WR motor neurons, numerous APP/Rab7-positive vesicles were observed which were mostly LC3-negative, suggesting they are not autophagosomes.ConclusionsWe conclude that endosomal APP/Rab7 staining reflects impaired vesicle trafficking in WR mouse motor neurons. Based on these findings human ALS tissues were analysed for APP in enlarged vesicles and were detected in spinal cord motor neurons in six out of fourteen sporadic ALS cases. These enlarged vesicles were not detected in any of the familial ALS cases. Thus our study provides the first evidence for wobbler-like aetiologies in human ALS and suggests that the genes encoding proteins involved in vesicle trafficking should be screened for pathogenic mutations.

Overshooting production of satellite cells in murine skeletal muscle affected by the mutation "muscular dystrophy with myositis" (mdm, Chr 2)

Abstract.The hereditary degenerative muscle disease ”muscular dystrophy with myositis” of the mouse (MDM, genotype mdm/mdm) is caused by a spontaneous recessive mutation on chromosome 2. Skeletal muscles of MDM mice degenerate during the first few postnatal weeks and subsequently regenerate. The cellular and molecular mechanisms of this muscle disease are not yet understood. An inflammatory component as implied by its name seems unlikely. By applying quantitative electron microscopy, we demonstrate a transient increase of up to 4-fold in the satellite cell frequency in both fast and slow muscles of affected animals. This difference from wildtype controls (+/+ or +/mdm) is also reflected in the yield of mononucleate myogenic cells upon dissociation of skeletal muscle and subsequent cell culture. Unlike the increase in satellite cell density in the myotonic ADR mouse, this phenomenon is not accompanied with a shift to more oxidative fibre types. It may however be related to the more profuse micro-vascularization of MDM muscle.

Increased density of satellite cells in the absence of fibre degeneration in muscle of myotonic mice

SummaryA mutant mouse with a hereditary myotonia, ‘arrested development of righting response’, ADR, was investigated with respect to mononucleated cell populations in skeletal muscle. Upon enzymatic dissociation of different muscles from mice aged between 15 and 120 days, a 3-to 5-fold higher yield of mononucleated cells per muscle fresh weight was obtained from mice with the ADR syndrome than from control mice. Clonal cell culture showed that the absolute number of cells with myogenic potential was increased and that mutant clones had shorter generation times than wild-type controls. Morphological differentiation of ADR myotubes was indistinguishable from that of the controls. Light microscopy confirmed the presence of increased numbers of mononucleated cells per muscle volume. At the ultrastructural level, there were 3.3 times as many satellite cells (the myogenic stem cells of mature muscle) per myofibre nucleus in ADR than in controls. Because no fibre degeneration was observed in the ADR mutant, we conclude that the enlarged mutant satellite cell pool is not a result of compensatory proliferation but is a consequence of fibre-type transformation and/or delayed maturation of the myotonic muscle.

Mutual interference of myotonia and muscular dystrophy in the mouse: a study on ADR-MDX double mutants

For Duchenne muscular dystrophy (DMD, dystrophin deficiency) and Thomsen/Becker myotonia (muscular chloride channel deficiency) genetically homologous mouse models are available, the dystrophin-deficient MDX mouse and the myotonic ADR mouse. Whereas the latter shows more severe symptoms than human myotonia patients, the MDX mouse, in contrast to DMD patients, is only mildly affected. We have introduced, by appropriate breeding, the defect leading to myotonia (Clc1 null mutation, adr allele) into MDX mice, thus creating ADR-MDX double mutants. The expectation was that, due to mechanical stress during myotonic cramps, the ADR status should symptomatically aggravate the muscle fibre necrosis caused by the dystrophin deficiency. The overall symptoms of the double mutants were dominated by myotonia. Weight reduction and premature death rate were higher in ADR-MDX than in ADR mice. Sarcolemmal ruptures as indicated by influx into muscle fibres of serum globulins and injected Evans blue were found with great inter-individual variation in MDX and in ADR-MDX muscles. Affected fibres were found mainly in large groups in MDX but single or in small clusters in ADR-MDX leg muscles. The symptoms of myotonia (aftercontractions, shift towards oxidative fibres) were less pronounced in ADR-MDX than in ADR muscles. Conversely, numbers of damaged fibres as well as the percentage of central nuclei (an indicator of fibre regeneration) were significantly lower in ADR-MDX than in MDX skeletal muscles. Thus it appears that, at the level of the muscle fibre, myotonia and muscular dystrophy attenuate each other.