Entorhinal cortex lesioning promotes neurogenesis in the hippocampus of adult mice.

Hippocampal neurogenesis in adult mammals is influenced by many factors. Lesioning of the entorhinal cortex is a standard model used to study injury and repair in the hippocampus. Here we use bromodeoxyuridine (BrdU) labeling combined with immunohistochemical identification using cell type specific markers to follow the fate of neural progenitors in the hippocampus following entorhinal cortex lesioning in mice. We show that unilateral entorhinal cortex lesioning does not alter the rate of neural progenitor proliferation in the ipsilateral dentate gyrus during the first 3 days after lesioning. However it enhances cell survival at 42 days post-lesioning leading to an increased number of beta-III tubulin and calbindin-immunoreactive neurons being produced. By contrast, when BrdU was administered 21 days post-lesioning, the number of surviving cells 21 days later was similar on the lesioned and non-lesioned sides. Thus, acutely entorhinal cortex lesioning promotes neurogenesis by enhancing survival of either neural progenitors or their progeny. However, this stimulus to neurogenesis is not sustained into the recovery period.

Transgenic rescue of Krabbe disease in the twitcher mouse.

The twitcher mouse is a natural model of Krabbe disease caused by galactocerebrosidase (GALC) deficiency. Previous attempts at rescuing the twitcher mouse by bone marrow transplantion, viral transduction, or transgenesis were only partially successful. Here, we report the transgenic (tg) rescue of the twitcher mouse with a BAC clone harboring the entire GALC. The twi/twi/hGALC tg mice exhibited growth, motor function, and fertility similar to those of nonaffected animals. These animals had normal levels of GALC activity in brain and were free of the typical twitcher demyelinating pathology. Surprisingly, GALC expression in twi/twi hGALC tg kidneys was low and galactocerebroside storage was only partially cleared. Nonetheless, these mice have been maintained for over 1 year without any sign of disease. Since pathological damage associated with GALC deficiency is confined to the nervous system, our work represents the first successful rescue of the twitcher mouse and opens the possibility of developing novel therapeutic approaches.

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease.

Canavan disease, an inherited leukodystrophy, is caused by mutations in the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups. Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. Of these, 14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAG-->TGG, X314W), and one splice acceptor site mutation (IVS1 - 2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1 - 2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frame shift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.

Molecular basis of late-life globoid cell leukodystrophy.

Globoid cell leukodystrophy is an autosomal recessive inherited disease caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Although the severe, rapidly progressing infantile form is the most common, late-onset forms have been described. We investigated the molecular basis of GALC deficiency in a patient with a late-life mild form of globoid cell leukodystrophy who survived into the eighth decade. Since material suitable for mutation analysis was no longer available from the proband, her GALC genotype was reconstructed by analyzing this gene in her six obligate carrier offspring. One allele contained the mutation 809G>A (G270D) in the 1637C background, while the other allele contained three sequence variants: 1609G>A (G537R), 1873G>A (A625T), and 1650T>A (V550V) in the 1637T background. These mutations were confirmed in the proband's genomic DNA isolated from a sural nerve biopsy. Expression studies indicated that the G537R is a disease-causing mutation, as it resulted in no GALC activity, either alone or together with the A625T. This A625T sequence variant did not affect the enzyme activity, at least when expressed in the 1637T background. The mild clinical phenotype was likely to be associated with the 809G>A, since residual GALC activity, about 17% of the control activity, was detected in the expression studies of this mutation. This mutation has been found in several other patients with late-onset GLD.

Molecular heterogeneity of late-onset forms of globoid-cell leukodystrophy.

Globoid-cell leukodystrophy (GLD) is an autosomal recessive inherited disorder caused by the deficiency of galactocerebrosidase, the lysosomal enzyme responsible for the degradation of the myelin glycolipid galactocerebroside. Although the most common form of the disease is the classical infantile form (Krabbe disease), later-onset forms also have been described. We have analyzed the galactocerebrosidase gene in 17 patients (nine families) with late-onset GLD and in 1 patient with classical Krabbe disease. Half of the patients were heterozygous for the large gene deletion associated with the 502C-->T polymorphism, the most common mutation in infantile patients. Several novel mutations that result in deficient galactocerebrosidase activity were also identified in these patients. They include the missense mutations R63H, G95S, M101L, G268S, Y298C, and I234T; the nonsense mutation S7X; a one-base deletion (805delG); a mutation that interferes with the splicing of intron 1; and a 34-nt insertion in the RNA, caused by the aberrant splicing of intron 6. All of these genetic defects are clustered in the first 10 exons of the galactocerebrosidase gene and therefore affect the 50-kD subunit of the mature enzyme. Studies on the distribution and enzymatic activity of the polymorphic alleles 1637T/C (I546/T546) provided support for previous data that had indicated the existence of two galactocerebrosidase forms with different catalytic activities in the general population. Our data also indicate that the mutations occur preferentially in the "low activity" 1637C allele.

A human kidney cDNA which induces a cell surface protein epitope recognized by a monoclonal antibody against galactosylceramide.

Antibodies against the myelin glycolipid galactosylceramide are widely used to study the distribution and function of this molecule. However, anti-galactosylceramide antibodies are not monospecific and have been shown to recognize epitopes carried not only by other glycolipids, but also by proteins. Using expression cloning we have identified a human kidney cDNA which induces a cell-surface protein recognized by the anti-galactosylceramide monoclonal antibody R-mab. These findings further support the idea that cross-reactive proteins may mediate some of the biological effects of the anti galactosylceramide antibodies.

Late-onset GM2 gangliosidosis: Ashkenazi Jewish family with an exon 5 mutation (Tyr180-->His) in the Hex A alpha-chain gene.

Late-onset GM2 gangliosidosis is a variant form of Tay-Sachs disease characterized by onset of symptoms and signs in adolescence or in early adult life. The deficiency of beta-hexosaminidase A (Hex A) in this form of GM2 gangliosidosis has been invariably associated with the presence of the Gly269-->Ser substitution in the alpha-chain. We found two siblings of Ashkenazi Jewish descent diagnosed with late-onset GM2 gangliosidosis who were negative for the Gly269-->Ser mutation. Analysis of the HEXA gene showed that they were compound heterozygotes for the functionally silent 4-bp insertion in exon 11, typical of the infantile form of the disease and for a novel mutation, T538-->C, resulting in the missense Tyr180-->His. Expression studies in COS-7 cells suggested that the effect of this mutation was to decrease the stability of the alpha-chain at physiologic temperatures and therefore to indirectly affect the formation of mature Hex A.

Intrathecal synthesis of anti-sulfatide IgG is associated with peripheral nerve disease in acquired immunodeficiency syndrome.

Peripheral nervous system involvement in the acquired immunodeficiency syndrome (AIDS) can take the form of an acute or chronic inflammatory demyelinating polyneuropathy, polyradiculopathy, mononeuropathy multiplex, or autonomic neuropathy. There is no widely held consensus on the etiology of PNS or other neurological complications associated with HIV infection. We report here that PNS disease in HIV-infected individuals is associated with intrathecal synthesis of an antibody directed against sulfatide, a major component of myelin. The anti-sulfatide antibody is also present nonspecifically in serum. The antibody requires the presence of the 3-O-sulfogalactosyl residue for binding and recognizes preferentially the hydroxy fatty acid-containing form of sulfatide. Anti-sulfatide antibodies are therefore one of the humoral factors responsible for demyelinating diseases in AIDS patients.

Correction of the galactocerebrosidase deficiency in globoid cell leukodystrophy-cultured cells by SL3-3 retroviral-mediated gene transfer.

Globoid cell leukodystrophy (GCL) or Krabbe disease is an autosomal recessive inherited disease caused by the deficiency of galactocerebrosidase, the lysosomal enzyme responsible for the degradation of galactocerebroside, a major component of myelin. An animal model homologue of GCL is the twitcher mouse. In the present work, using novel recombinant retroviruses harboring the SL3-3 LTR, we have been able to stably correct the galactocerebrosidase deficiency in twitcher mouse TM-2 cells and in primary human fibroblasts from a patient with globoid cell leukodystrophy. These results show the possibility of retroviral-mediated gene therapy for the treatment of GCL.

Substitution of alanine543 with a threonine residue at the carboxy terminal end of the beta-chain is associated with thermolabile hexosaminidase B in a Jewish family of Oriental ancestry.

Thermolabile forms of the lysosomal enzyme beta-hexosaminidase B (Hex B), likely to result from different genetic defects, have been described. Ten individuals in five generations of a family of Oriental Jewish ancestry were identified biochemically as carriers of a thermolabile Hex B form. The beta-chain thermolability was found to be associated with the presence of a G --> A transition at nucleotide 1627 of the HEX B gene causing the substitution of Ala543 with a threonine. Oriental Jew whose Hex B was heat labile. Since thermolabile Hex B has been shown to occur more frequently among Jews of Oriental origin, the Ala543 --> Thr mutation may be the common mutation associated with beta-chain thermolability in this ethnic group.

Comparison of digitized ECGs simultaneously recorded with CR and V leads.

Evidence is presented that electrocardiograms recorded with bipolar chest-right arm (CR) leads are diagnostically similar to electrocardiograms recorded with unipolar V leads. Electrocardiograms were simultaneously recorded with CR and V leads on six chest sites in 45 cardiac patients and submitted, unmarked, for evaluation, by four cardiologists. In spite of relatively small differences in the amplitudes of P, Q, R, S, and T waveforms, the diagnosis based on tracings recorded with CR leads was similar to the diagnosis based on tracings recorded with V leads in nearly 90% of patients. Because CR leads are set up with only two electrodes, one on the right arm and one on the chest, their use in cardiac emergencies saves time and simplifies the recording technique. This investigation is part of a project aimed at developing a portable electrocardiograph for use outside the hospital or clinic.

Substrate specificity of human liver neutral alpha-mannosidase.

The digestion of radiolabelled natural oligosaccharide substrates by human liver neutral alpha-mannosidase has been studied by h.p.l.c. and h.p.t.l.c. The high-mannose oligosaccharides Man9GlcNAc and Man8GlcNAc are hydrolysed by the enzyme by two distinct non-random routes to a common product of composition Man6GlcNAc, which is then slowly converted into a unique Man5GlcNAc oligosaccharide, Man alpha(1----2)Man alpha(1----2)Man alpha(1----3)[Man alpha (1----6)] Man beta(1----4)GlcNAc. These pathways are different from the processing and lysosomal catabolic pathways for these structures. In particular, the alpha(1----2)-linked mannose residues attached to the core alpha(1----3)-linked mannose residue are resistant to hydrolysis. The key processing intermediate, Man alpha(1----3)[Man alpha(1----6)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc, is not produced in the digestion of high-mannose glycans by the neutral alpha-mannosidase, but it is hydrolysed by the enzyme by a non-random route to Man beta(1----4)GlcNAc via the core structure Man alpha(1----3)[Man alpha(1----6)]Man beta(1----4)GlcNAc. In contrast with its ready hydrolysis by lysosomal alpha-mannosidase, the core alpha(1----3)-mannosidic linkage is quite resistant to hydrolysis by neutral alpha-mannosidase. The precise specificity of neutral alpha-mannosidase towards high-mannose oligosaccharides suggests that it has a role in the modification of such structures in the cytosol.

Substrate specificity of the bovine and feline neutral alpha-mannosidases.

Neutral alpha-mannosidases were prepared from bovine and cat liver. The activities were distinguished from lysosomal and Golgi alpha-mannosidases by their neutral pH optima, relatively low Km for their synthetic substrate p-nitrophenyl alpha-D-mannoside, inhibition by Zn2+ and absence of inhibition by Co2+, EDTA, low concentrations of swainsonine, or deoxymannojirimycin. The cytosolic alpha-mannosidases were not retained by concanavalin A-Sepharose. They were able to degrade efficiently a variety of oligosaccharides with structures corresponding to certain high-mannose glycans or the oligomannosyl parts of hybrid and complex glycans. However, unlike lysosomal alpha-mannosidases from the same species these enzymes were not able to degrade Man9GlcNAc2 efficiently, and the bovine neutral alpha-mannosidase was not able to degrade a hexasaccharide with a structure analogous to Man5GlcNAc2-PP-dolichol. Sharp differences were noted for the bovine and cat enzymes with regard to the specificity of degradation. The bovine neutral alpha-mannosidase degraded the substrates by defined pathways, but the cat neutral alpha-mannosidase often produced complex mixtures of products, especially from the larger oligosaccharides. Therefore the bovine enzyme resembled the rat and human cytosolic alpha-mannosidases, but the cat enzyme did not. The bovine and cat neutral alpha-mannosidases, unlike the corresponding lysosomal activities, did not show specificity for the hydrolysis of the (1----3)- and (1----6)-linked mannose residues in the N-linked glycan pentasaccharide core.

A human lysosomal alpha(1----6)-mannosidase active on the branched trimannosyl core of complex glycans.

Normal human fibroblasts and fibroblasts from a patient with alpha-mannosidosis were grown in the presence or absence of 100 microM swainsonine for 7 days. Accumulated oligosaccharides were isolated and analysed by high performance liquid chromatography (HPLC) and methylation analysis. Man alpha 1----3Man beta 1----4GlcNAc and Man alpha 1----2Man alpha 1----3-Man beta 1----4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised greater than 80% of the total oligosaccharides in untreated mannosidosis cells. However, Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc was the major Man3GlcNAc isomer present after 7 days of swainsonine treatment. No mannose-containing oligosaccharides were detected in control fibroblasts in the absence of swainsonine but, in its presence, oligosaccharides containing 2-9 mannose residues accumulated. Man alpha 1----6[Man alpha 1----3]-Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc and Man alpha 1----6-[Man alpha 1----3]Man beta 1----4GlcNAc were the major components (67%). Surprisingly, Man alpha 1----3Man beta 1----4GlcNAc was only observed in swainsonine-treated control cells during the recovery period after removal of swainsonine. These studies suggest the presence of a second lysosomal alpha-mannosidase activity which is unaffected in genetic alpha-mannosidosis, but is inhibited by swainsonine. This enzyme would cleave the alpha(1----6)-linked mannose residue from branched Man3GlcNAc to form Man alpha 1----3Man beta 1----4GlcNAc. To confirm this hypothesis, fractions from alpha-mannosidosis and control fibroblasts that bound to concanavalin A (ConA)-Sepharose and were eluted with 0.5 M alpha-methyl mannoside were incubated at pH 4.0 with Man alpha 1----6[Man alpha 1----3]Man beta 1----4-GlcNAc. As anticipated, Man alpha 1----3Man beta 1----4GlcNAc was the sole product using enzyme from mannosidosis fibroblasts, while the major product from control fibroblasts was Man alpha 1----6Man beta 1----4GlcNAc. This confirmed the presence of a swainsonine-inhibitable alpha(1----6)-mannosidase activity unaffected by the disease. The differing substrate specificities of the alpha(1----6)-mannosidase and the major lysosomal alpha-mannosidase indicate that the alpha(1----6)-mannosidase plays an important role in the generation of the oligosaccharides accumulated in alpha-mannosidosis patients.

A human lysosomal alpha-mannosidase specific for the core of complex glycans.

A novel lysosomal alpha-mannosidase, with unique substrate specificity, has been partially purified from human spleen by chromatography through concanavalin A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. This enzyme can catalyze the hydrolysis of only 1 mannose residue, that which is alpha(1----6)-linked to the beta-linked mannose in the core of N-linked glycans, as found in the oligosaccharides Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc and Man alpha(1----6)Man beta(1----4) GlcNAc. The newly described alpha-mannosidase does not catalyze the hydrolysis of mannose residues outside of the core, even if they are alpha(1----6)-linked, and is not active on the other alpha-linked mannose in the core, which is (1----3)-linked. The narrow specificity of the novel mannosidase contrasts sharply with that of the major lysosomal alpha-mannosidase, which is able to catalyze the degradation of oligosaccharides containing diverse linkage and branching patterns of the mannose residues. Importantly, although the major mannosidase readily catalyzes the hydrolysis of the core alpha(1----3)-linked mannose, it is poorly active towards the alpha(1----6)-linked mannose, i.e. the very same mannose residue for which the newly characterized mannosidase is specific. The novel enzyme is further differentiated from the major lysosomal alpha-mannosidase by its inability to catalyze the efficient hydrolysis of the synthetic substrate p-nitrophenyl alpha-mannoside, and by the strong stimulation of its activity by Co2+ and Zn2+. Similarly to the major mannosidase, it is strongly inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, but not by deoxymannojirimycin. The presence of this novel alpha-mannosidase activity in human tissues provides the best explanation, to date, for the structures of the oligosaccharides stored in human alpha-mannosidosis. In this condition the major lysosomal alpha-mannosidase activity is severely deficient, but apparently the alpha(1----6)-mannosidase is unaffected, so that the oligosaccharide structures reflect the unique specificity of this enzyme.

Digitized cardiac potentials recorded with CR leads. Development of a portable electrocardiograph.

This report describes the advantages of recording cardiac potentials in digital rather than in analog form and of using statistical methods that compare a patient's measurements with values measured in a normal population. In this study, expansion of the time axis in digitized electrocardiograms was used to accurately determine the moments when the Q, R, and S waves began and ended. This work is part of a plan to develop a portable electrocardiograph that could be available to physicians at all times. The immediate availability of such an instrument could shorten the time required to reach a diagnosis and institute treatment in cardiac emergencies occurring where diagnostic facilities are unavailable.

The substrate-specificity of human lysosomal alpha-D-mannosidase in relation to genetic alpha-mannosidosis.

The specificity of human liver lysosomal alpha-mannosidase (EC 3.2.1.24) towards a series of oligosaccharide substrates derived from high-mannose, complex and hybrid asparagine-linked glycans and from the storage products in alpha-mannosidosis was investigated. The enzyme hydrolyses all alpha(1-2)-, alpha(1-3)- and alpha(1-6)-mannosidic linkages in these glycans without a requirement for added Zn2+, albeit at different rates. A major finding of this study is that all the substrates are hydrolysed by non-random pathways. These pathways were established by determining the structures of intermediates in the digestion mixtures by a combination of h.p.t.l.c. and h.p.l.c. before and after acetolysis. The catabolic pathway for a particular substrate appears to be determined by its structure, raising the possibility that degradation occurs by an uninterrupted sequence of steps within one active site. The structures of the digestion intermediates are compared with the published structures of the storage products in mannosidosis and of intact asparagine-linked glycans. Most but not all of the digestion intermediates derived from high-mannose glycans have structures found in intact asparagine-linked glycans of human glycoproteins or among the storage products in the urine of patients with mannosidosis. However, the relative abundances of these structures suggests that the catabolic pathway is not the same as the processing pathway. In contrast, the intermediates formed from the digestion of oligosaccharides derived from hybrid and complex N-glycans are completely different from any processing intermediates and also from the oligosaccharides of composition Man2-4GlcNAc that account for 80-90% of the storage products in alpha-mannosidosis. It is postulated that the structures of these major storage products arise from the action of an exo/endo-alpha(1-6)-mannosidase on the partially catabolized oligomannosides that accumulate in the absence of the main lysosomal alpha-mannosidase.

Application of lectin histochemistry and carbohydrate analysis to the characterization of lysosomal storage diseases.

In lysosomal storage diseases that involve a defect in the catabolism of glycoconjugates, lectin histochemistry adds a new dimension to the characterization of stored carbohydrates as it identifies sugar residues in situ in the affected cells and, thus, determines which cell types are affected by storage. It may be combined with chemical and biochemical analysis by h.p.l.c. The present review summarizes recent results for a variety of storage diseases and presents new data for GM1-gangliosidosis.