Harald Jockusch

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.

A Role for Polyproline Motifs in the Spinal Muscular Atrophy Protein SMN PROFILINS BIND TO AND COLOCALIZE WITH SMN IN NUCLEAR GEMS

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of α-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.

The Metalloprotease Disintegrin ADAM8 PROCESSING BY AUTOCATALYSIS IS REQUIRED FOR PROTEOLYTIC ACTIVITY AND CELL ADHESION

ADAMs (a disintegrinand metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu330 to Gln exchange (EQ-A8) in the Zn2+ binding motif (HE330LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC50∼50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.

Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts.

Tissue engineering of skeletal muscle from cultured cells has been attempted using a variety of synthetic and natural macromolecular scaffolds. Our study describes the application of artificial scaffolds (collagen sponges, CS) consisting of collagen‐I with parallel pores (width 20–50 μm) using the permanent myogenic cell line C2C12. CS were infiltrated with a high‐density cell suspension, incubated in medium for proliferation of myoblasts prior to further culture in fusion medium to induce differentiation and formation of multinucleated myotubes. This resulted in a parallel arrangement of myotubes within the pore structures. CS with either proliferating cells or with myotubes were grafted into the beds of excised anterior tibial muscles of immunodeficient host mice. The recipient mice were transgenic for enhanced green fluorescent protein (eGFP) to determine a host contribution to the regenerated muscle tissue. Histological analysis 14–50 days after surgery showed that donor muscle fibres had formed in situ with host contributions in the outer portions of the regenerates. The function of the regenerates was assessed by direct electrical stimulation which resulted in the generation of mechanical force. Our study demonstrated that biodegradable CS with parallel pores support the formation of oriented muscle fibres and are compatible with force generation in regenerated muscle.

Wobbler, a mutation affecting motoneuron survival and gonadal functions in the mouse, maps to proximal chromosome 11

The wobbler mouse (genotype wr/wr) has been considered as an animal model for human neurodegenerative disorders. In the homozygous condition, the autosomal mutation wobbler (wr) causes a motoneuron disease and gonadal dysfunction. We have genetically mapped the wr gene, using an interspecific backcross between the laboratory strain C57BL/6J (wr/+) and Mus spretus. The expected percentage of wobbler progeny were obtained, but heterogeneous expression of the wobbler phenotype indicated the existence of modifier genes in the M. spretus genetic background. The segregation of DNA markers of known chromosomal location among wobbler progeny and unaffected mice was scored. Close linkage of wr was obtained with Erbb and Rel on chromosome 11 and the gene order cen-Nfh-Erbb-wr-Rel-Hba-Il-3 was established. Closely linked markers like Erbb provide tools for a prognostic DNA diagnosis of the wobbler disease, and thereby for its analysis by descriptive and experimental embryology.

Slime mould actin: Homology to vertebrate actin and presence in the nucleus

Abstract Sequence homology between muscle actin of a mammal (rabbit) and cytoplasmic actin of a slime mould ( Physarum polycephalum ) is revealed by tryptic finger-printing. The same technique was used to demonstrate actin in the nucleus of Physarum .

Two mutants of tobacco mosaic virus temperature-sensitive in two different functions

Abstract A method is described of estimating the approximate quantities of native and denatured TMV coat proteins in infected tobacco leaves. It consists of extracting the leaf tissue with neutral buffer, separating soluble and insoluble fractions by low speed centrifugation, dissolving the insoluble fractions with acetic acid, and analytical polyacrylamide gel electrophoresis in the presence of 8 M urea. This method and several biological tests were applied to two temperature-sensitive mutants of TMV, one of which is a coat protein (ts-I) mutant while the other is defective in propagation of the infective principle (ts-II). At high temperature the ts-I mutant produces large amounts of denatured coat protein polypeptide chains and has the ability to inhibit multiplication of superinfecting wild type TMV, while the ts-II mutant does not exert these functions.

Temperatursensitive Mutanten des Tabakmosaikvirus

A. Voraussetzungen I. Strukturen, die aus helixartig angeordneten globul/iren Proteinuntereinheiten bestehen, sind bei Tieren, Pflanzen, Bakterien und Viren verbreitet. Ihre Zerlegung und I~ekonstitution augerhalb der Zelle dureh geeignete Ver~nderungen des physikochemischen Milieus ist in mehreren F/illen gelungen (z.B. S z ~ GYS~Gu 1951; AUDLEY und COCKBAI~, 1966; AB~AM und KOFFLEn, 1963; POOLAZOV, BO~RSEmUS und BELAV~SnVA, 1965). Im Falle des Tabakmosaikvirus bildet das gesamte Virusteilehen eine solche Struktur, die exakt oder angens ~us alkali-abgebautem Via-us (Sc~AMM, 1947), nukleins~urefreiem Hfillprotein allein (TAKAttASHI und ISHn, 1952) oder zusammen mit intakter RNS (FRAENKELCO~AT und WILLIAMS, 1955) wieclerhergestellt werden konnte. Die Desaggregationsund Reaggregationsversuehe zeigen, dag diese Proteiniiberstrukturen nieht durch kovalente Bindungen zusammengehalten werden und dab die Eigensehaften der Untereinheiten ihre geometrisehe Anordnung bestimmen. Die F~higkeit der Untereinheiten, geordnete Aggregate zu bilden, kann ebenso wie die katalytische Aktivits yon Enzymen dureh Einfliisse, die kovalente Bindungen nicht angreifen, zerstSrt werden (Denaturierung -Def in i t ion vgl. JoLY, 1965). Im Falle des TMV wurde dutch Renaturierung des phenolund harnstoffdenaturierten HP wahrscheinlich gemacht, dag die Aminoss allein die Faltung der Untereinheiten und damit weitgehend die morphologische Struktur des Virusteilchens bestimmt (AI~DElCEtt, 1959). Das t IP des TMV ist sowohl strukturell (Zusammenfassung z. B. : CASPA~, 1963) als auch proteinchemiseh (Zusammenfassung z. ]3. : ANDEI~EI~, 1963) das bestuntersuch~e Strukturprotein. Dureh Untersuchungen fiber den genetisehen Code wurden Mutanten mit einze]nen oder wenigen bekannten Aminosgureaustausehen zuggnglieh (~u 1962, 1964; TSVGITA und F~AE~KEL-CoN~AT, 1962). Bei physiologisehen Versuehen (JocKCSCH, 1966a) wurde gefunden, dab viele der Nitr i tmutanten WIT~r~A~s und einige Spontanmutanten des TMV (M~C~E~S, i942 und unpubh) temperatursensitiv (ts) sind: im Gegensatz zum Wfldtyp und temperaturresistenten (tr) Mutanten produzieren sie bei 30--32 ~ C in der Wirtspflanze sehr viel weniger extrahierbares, infekti6ses Virus als bei 23 ~ C (JocKgscI~, 196~). Eine parallele Beobaehtung hat Kassa~IS (1957) am Tomaten-Aueubamosaikvirus, einer natfirliehen Variante des TMV,

Spatiotemporal progression of neurodegeneration and glia activation in the wobbler neuropathy of the mouse

The wobbler mouse (phenotype WR; genotype wr/wr) has been investigated as a model for neurodegenerative diseases like SMA and ALS. A new diagnostic marker based on a polymorphism in the closely linked chaperonine gene Cct4 enabled us to diagnose the allelic status at the wr locus within the original background strain C57BL/6. Using this marker, we investigated the spatiotemporal progression of neuropathology in WR mice from postnatal day (d.p.n.) 10 to 60. Neurodegeneration starts at 13 d.p.n. in the thalamus (N. ventralis), in deep cerebellar nuclei, brain stem (N. vestibularis) and spinal cord interneurons. The motor nuclei of spinal nerves and motoneurons degenerate from 15 d.p.n. onward. Reactive astrocytes are observed around 17 d.p.n. in the white and grey matter of the spinal cord. Microgliosis occurs only from 23 d.p.n. onward. Our data demonstrate that in the WR disease, neurodegeneration in thalamus, cerebellum, and brain stem precedes motoneuron degeneration, astrogliosis and microgliosis.

Localization of GFP in Frozen Sections from Unfixed Mouse Tissues: Immobilization of a Highly Soluble Marker Protein by Formaldehyde Vapor

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic “green mice” and for a transplantation experiment.