Expression of myostatin pro domain results in muscular transgenic mice.

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Depression of myostatin activity leads to increased muscle growth and carcass lean yield. In an attempt to down-regulate myostatin, transgenic mice were produced with a ribozyme-based construct or a myostatin pro domain construct. Though the expression of the ribozyme was detected, muscle development was not altered by the ribozyme transgene. However, a dramatic muscling phenotype was observed in transgenic mice carrying the myostatin pro domain gene. Expression of the pro domain transgene at 5% of beta-actin mRNA levels resulted in a 17-30% increase in body weight (P < 0.001). The carcass weight of the transgenic mice showed a 22-44% increase compared with nontransgenic littermates at 9 weeks of age (16.05 +/- 0.67 vs. 11.16 +/- 0.28 g in males; 9.99 +/- 0.38 vs. 8.19 +/- 0.19 g in females, P < 0.001). Extreme muscling was present throughout the whole carcass of transgenic mice as hind and fore limbs and trunk weights, all increased significantly (P < 0.001). Epididymal fat pad weight, an indicator of body fat, was significantly decreased in pro domain transgenic mice (P < 0.001). Analysis of muscle morphology indicated that cross-sectional areas of fast-glycolytic fibers (gastrocnemius) and fast-oxidative glycolytic fibers (tibialis) were larger in pro domain transgenic mice than in their controls (P < 0.01), whereas fiber number (gastrocnemius) was not different (P > 0.05). Thus, the muscular phenotype is attributable to myofiber hypertrophy rather than hyperplasia. The results of this study suggest that the over-expression of myostatin pro domain may provide an alternative to myostatin knockouts as a means of increasing muscle mass in other mammals.

Co-expression of multiple transgenes in mouse CNS: a comparison of strategies.

The introduction of two transgenes into one animal is increasingly common as transgenic experiments become more sophisticated. In this study we examine two strategies for creating double transgenic founders from a single microinjection. In the first approach, two constructs, each with its own promoter element, were coinjected into the pronucleus. In the second approach, both transgenes were cloned into one vector, separated by an internal ribosomal entry site (IRES), and placed under control of a single promoter. Both strategies save time and increase the percentage of double transgenic offspring over the standard method of mating single transgenic lines. However, despite high transgene copy numbers, the bicistronic lines did not show robust expression of either protein. Copy number and protein expression correlated much better in the coinjected lines, with expression levels in one line approaching that observed in some of our best single transgenic controls. Thus we recommend coinjection of individual plasmids for the generation of multiply transgenic founders.

Variation in the biochemical/biophysical properties of mutant superoxide dismutase 1 enzymes and the rate of disease progression in familial amyotrophic lateral sclerosis kindreds.

Mutations in superoxide dismutase 1 (SOD1) polypeptides cause a form of familial amyotrophic lateral sclerosis (FALS). In different kindreds, harboring different mutations, the duration of illness tends to be similar for a given mutation. For example, patients inheriting a substitution of valine for alanine at position four (A4V) average a 1.5 year life expectancy after the onset of symptoms, whereas patients harboring a substitution of arginine for histidine at position 46 (H46R) average an 18 year life expectancy after disease onset. Here, we examine a number of biochemical and biophysical properties of nine different FALS variants of SOD1 polypeptides, including enzymatic activity (which relates indirectly to the affinity of the enzyme for copper), polypeptide half-life, resistance to proteolytic degradation and solubility, in an effort to determine whether a specific property of these enzymes correlates with clinical progression. We find that although all the mutants tested appear to be soluble, the different mutants show a remarkable degree of variation with respect to activity, polypeptide half-life and resistance to proteolysis. However, these variables do not stratify in a manner that correlates with clinical progression. We conclude that the basis for the different life expectancies of patients in different kindreds of sod1-linked FALS may result from an as yet unidentified property of these mutant enzymes.

Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin.

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by the expansion of a glutamine repeat in the N-terminus of the huntingtin protein. To gain insight into the pathogenesis of HD, we generated transgenic mice that express a cDNA encoding an N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.

A role for serotonin in the circadian system revealed by the distribution of serotonin transporter and light-induced Fos immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet.

Components of the circadian system, the suprachiasmatic nucleus and the intergeniculate leaflet receive serotonin input from the raphe nuclei. Manipulations of serotonin neurotransmission disrupt cellular, electrophysiological, and behavioural responses of the circadian system to light, suggesting that serotonin plays a modulatory role in photic regulation of circadian rhythms. To study the relation between serotonin afferents and light-activated cells in the suprachiasmatic nucleus and intergeniculate leaflet, we used immunostaining for the serotonin transporter and for the transcription factor, Fos. Serotonin transporter, a plasma membrane protein located on serotonin neurons, regulates the amount of serotonin available for neurotransmission by re-accumulating released serotonin into presynaptic neurons; expression of Fos in the suprachiasmatic nucleus identifies light-activated cells involved in photic resetting of circadian clock phase. In the suprachiasmatic nucleus, immunostaining for serotonin transporter revealed a dense plexus of fibres concentrated primarily in the ventrolateral region. In the intergeniculate leaflet, serotonin transporter immunostaining identified vertically-oriented columns of fibres. Serotonin transporter immunostaining was abolished by pretreatment with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Exposure to light for 30 min during the dark phase of the light cycle induced Fos expression in the ventrolateral suprachiasmatic nucleus and intergeniculate leaflet regions. In both structures the Fos-expressing cells were encircled by serotonin transporter-immunoreactive fibres often in close apposition to these cells. These results support the idea that serotonin activity plays a modulatory role in processing of photic information within the circadian system.

Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins.

Missense mutations in two related genes, termed presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer's disease (FAD) pedigrees. In a variety of experimental in vitro and in vivo settings, FAD-linked presenilin variants influence the processing of the amyloid precursor protein (APP), leading to elevated levels of the highly fibrillogenic Abeta1-42 peptides that are preferentially deposited in the brains of Alzheimer Disease (AD) patients. In this report, we demonstrate that transgenic animals that coexpress a FAD-linked human PS1 variant (A246E) and a chimeric mouse/human APP harboring mutations linked to Swedish FAD kindreds (APP swe) develop numerous amyloid deposits much earlier than age-matched mice expressing APP swe and wild-type Hu PS1 or APP swe alone. These results provide evidence for the view that one pathogenic mechanism by which FAD-linked mutant PS1 causes AD is to accelerate the rate of beta-amyloid deposition in brain.

Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors.

Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for "replacement" we expressed familial Alzheimer's disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).

Endoproteolytic processing and stabilization of wild-type and mutant presenilin.

Presenilin 1 (PS1), mutated in pedigrees of early-onset familial Alzheimer's disease, is a polytopic integral membrane protein that is endoproteolytically cleaved into 27-kDa N-terminal and 17-kDa C-terminal fragments. Although these fragments are the principal PS1 species found in normal mammalian brain, the role of endoproteolysis in the maturation of PS1 has been unclear. The present study, which uses stably transfected mouse neuroblastoma N2a cells, demonstrates that full-length polypeptides, derived from either wild-type or A246E FAD-mutant human (hu) PS1, are relatively short-lived (t1/2 1.5 h) proteins that give rise to the N- and C-terminal PS1 fragments, which are more stable (t1/2 approximately 24 h). N-terminal fragments, generated artificially by engineering a stop codon at amino acid 306 (PS1-306) of wild-type huPS1, were short-lived, whereas an FAD-linked variant that lacked exon 9 (DeltaE9) and was not endoproteolytically cleaved exhibited a long half-life. These observations suggest that endoproteolytic cleavage and stability are not linked, leading us to propose a model in which wild-type full-length huPS1 molecules are first stabilized then subsequently endoproteolytically cleaved to generate the N- and C-terminal fragments. These fragments appear to represent the mature and functional forms of wild-type huPS1.

Hyperaccumulation of FAD-linked presenilin 1 variants in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.

Carrier-mediated serotonin release induced by d-fenfluramine: studies with human neuroblastoma cells transfected with a rat serotonin transporter.

The NMB human neuronal cell line, transfected with a newly prepared plasmid expressing rat serotonin transporter (NMB-rSERT), shows specific [3H]5-HT uptake which is blocked by citalopram and fenfluramine (F) stereoisomers with IC50 values (1 nM. 0.5 microM (dF) and and 5 microM (IF), respectively) which are similar to those found in rat brain synaptosomes. d-Fenfluramine (0.5 and 10 microM) also stimulates tritium release from NMB-rSERT cells preloaded with [3H-]-5-HT. The d-fenfluramine-induced [3H-]5-HT release is blocked by 0.3 microM citalopram and is dependent on the density of SERT expressed per cell, but is not affected by removal of Ca++ ions from the incubation medium. Manipulation of the Na+ gradient across the plasma membrane (replacing 60 mM NaCl with an equimolar concentration of KCl or choline) also induced [3H-]5-HT release from NMB-rSERT cells, which was inhibited by 0.3 microM citalopram. These results, together with the finding that NMB-rSERT cells preloaded with 500 nM unlabelled 5-HT take up [3H-]d-fenfluramine, make NMB-rSERT cells a valuable tool for studying the transporter-mediated exchange release induced by amphetamine derivatives.

Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acids antisense to the dopamine transporter.

Human neuroblastoma NMB cells take up [3H]dopamine in a selective manner indicating that dopamine transporters are responsible for this uptake. These cells were therefore used as a model to study dopamine neurotoxicity, and to elucidate the role of dopamine transporters in controlling cell death. Treatment with 0.05 0.4 mM dopamine changed cells' morphology within 4 h, accompanied by retraction of processes, shrinkage, apoptosis-like atrophy, accumulation of apoptotic particles, DNA fragmentation and cell death. Cycloheximide inhibited dopamine's effect suggesting that induction of apoptosis by dopamine was dependent upon protein synthesis. Dopamine cytotoxicity, monitored morphologically by flow cytometric analysis, and by lactate dehydrogenase released, was blocked by cocaine but not by the noradrenaline and serotonin uptake blockers desimipramine and imipramine, respectively. Attempting to inhibit dopamine transport and toxicity in a drug-free and highly selective way, three 18-mer dopamine transporter antisense phosphorothioate oligonucleotides (numbers 1, 2 and 3) and a new plasmid vector expressing the entire rat dopamine transporter complementary DNA in the antisense orientation were prepared and tested. Antisense phosphorothioate oligonucleotide 3 inhibited [3H]dopamine uptake in a time- and dose-dependent manner. Likewise, transient transfection of NMB cells with the plasmid expressing dopamine transporter complementary DNA in the antisense orientation partially blocked [3H]dopamine uptake. Antisense phosphorothioate oligonucleotide 3 also decreased, dose-dependently, the toxic effect of dopamine and 6-hydroxydopamine. Western blot analysis with newly prepared anti-human dopamine transporter antibodies showed that antisense phosphorothioate oligonucleotide 3 decreased the transporter protein level. These studies contribute to better understand the mechanism of dopamine-induced apoptosis and neurotoxicity.