Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice.

It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.

Inhibition of cardiac delayed rectifier K+ current by overexpression of the long-QT syndrome HERG G628S mutation in transgenic mice.

Mutations in the HERG gene are linked to the LQT2 form of the inherited long-QT syndrome. Transgenic mice were generated expressing high myocardial levels of a particularly severe form of LQT2-associated HERG mutation (G628S). Hearts from G628S mice appeared normal except for a modest enlargement seen only in females. Ventricular myocytes isolated from adult wild-type hearts consistently exhibited an inwardly rectifying E-4031-sensitive K+ current resembling the rapidly activating cardiac delayed rectifier K+ current (Ikr) in its time and voltage dependence; this current was not found in cells isolated from G628S mice. Action potential duration was significantly prolonged in single myocytes from G628S ventricle (cycle length=1 second, 26 degrees C) but not in recordings from intact ventricular strips studied at more physiological rates and temperature (200 to 400 bpm, 37 degrees C). ECG intervals, including QT duration, were unchanged, although minor aberrancies were noted in 20% (16/80) of the G628S mice studied, primarily involving the QRS complex and, more rarely, T-wave morphology. The aberrations were more commonly observed in females than males but could not be correlated with sex-based differences in action potential duration. These results establish the presence of IKr in the adult mouse ventricle and demonstrate the ability of the G628S mutation to exert a dominant negative effect on endogenous IKr in vivo, leading to the expected LQT2 phenotype of prolonged repolarization at the single cell level but not QT prolongation in the intact animal. The model may be useful in dissecting repolarization currents in the mouse heart and as a means of examining the mechanism(s) by which the G628S mutation exerts its dominant negative effect on native cardiac cells in vivo.

Dde-I restriction endonuclease fragmentation: a novel method of generating cDNA probes for in situ hybridization in brain.

We present a novel procedure for detection of low- and high-abundance messenger RNAs in the brain by in situ hybridization histochemistry, by using fragmented double-stranded cDNA as molecular probes. The procedure involves digesting the cDNA of interest with the restriction endonuclease from Desulfocibrio desulfuricans (Dde I digestion), followed by random primed labeling, which generates a family of high specific activity cDNA fragments. This procedure is a rapid, straightforward, and reproducible method of obtaining sensitive probes for in situ hybridization and is generally applicable to the analysis of the expression of a large number of genes. Here we report the use of this procedure to prepare probes for the detection of synapsin I, p150Glued, neurotensin, c-fos, and c-jun mRNAs in brain, using both isotopic and non-isotopic labeling methods. Because this procedure does not require complex recombinant DNA manipulations or oligonucleotide design, it should prove useful to the non-molecular biologist examining the expression of genes in the central nervous system.

Mutant and native human beta-amyloid precursor proteins in transgenic mouse brain.

Human beta-amyloid precursor protein (beta APP) has been targeted to transgenic neurons using synapsin I promoter-based chimeric transgenes. Native human beta APP was introduced as well as beta APP containing mutations genetically linked to familial Alzheimer's disease (AD) and to hereditary cerebral hemorrhage with amyloidosis-Dutch type. In mouse brain, human beta APP RNA was up to 60% as abundant as total endogenous beta APP RNA. Human beta APP gene expression was most abundant in the CA subfields of the hippocampus and in the piriform cortex. Correct processing of human beta APP at the beta-secretase cleavage site was demonstrated in transgenic mouse brains. Despite a 40% increase in total beta APP immunoreactivity in lines expressing mutant human beta APP, no evidence of amyloid deposition was found in brains of mice up to 14 months in age. Higher levels of mutant human beta APP, increased age, or other factors may be necessary to elicit beta-amyloid-related neuropathologies in the rodent brain.

Temporal onset of synapsin I gene expression coincides with neuronal differentiation during the development of the nervous system.

Synapsin I is the best characterized member of a family of nerve terminal-specific phosphoproteins implicated in the regulation of neurotransmitter release. During development, the expression of synapsin I correlates temporally and topographically with synapse formation, and recent physiological studies (Lu et al. [1992] Neuron 8:521-529.) have suggested that synapsin I may participate in the functional maturation of synapses. To better understand the temporal relationship between synapsin I gene expression and particular cellular events during neuronal development, we have used in situ hybridization histochemistry to localize synapsin I mRNA throughout the rat central and peripheral nervous systems during embryonic and postnatal development. From the earliest embryonic time points assayed (E12), the expression of the synapsin I gene was detectable in both the central and peripheral nervous systems. While, in general, levels of synapsin I mRNAs were high in utero, synapsin I cDNA probes revealed specific patterns of hybridization in different regions of the embryonic nervous system. To determine precisely the temporal onset of expression of the synapsin I gene during neuronal development, we examined in detail the appearance of synapsin I mRNA during the well characterized postnatal development of granule cells of the rat cerebellum and hippocampus. In both regions, the onset of synapsin I gene expression correlated with the period of stem cell commitment to terminal differentiation. Finally, our data demonstrate that, in a second phase, synapsin I gene expression increases to a maximum for a given neuronal population during a particular phase of differentiation, i.e., synaptogenesis.

The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1).

The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. We have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776 nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors.

Dynamics of synapsin I gene expression during the establishment and restoration of functional synapses in the rat hippocampus.

Synapse development and injury-induced reorganization have been extensively characterized morphologically, yet relatively little is known about the underlying molecular and biochemical events. To examine molecular mechanisms of synaptic development and rearrangement, we looked at the developmental pattern of expression of the neuron-specific gene synapsin I in granule cell neurons of the dentate gyrus and their accompanying mossy fibers during the main period of synaptogenic differentiation in the rat hippocampus. We found a significant difference between the temporal expression of synapsin I messenger RNA in dentate granule somata and the appearance of protein in their mossy fiber terminals during the postnatal development of these neurons. Next, to investigate the regulation of neuron-specific gene expression during the restoration of synaptic contacts in the central nervous system, we examined the expression of the synapsin I gene following lesions of hippocampal circuitry. These studies show marked changes in the pattern and intensity of synapsin I immunoreactivity in the dendritic fields of dentate granule cell neurons following perforant pathway transection. In contrast, changes in synapsin I messenger RNA expression in target neurons, and in those neurons responsible for the reinnervation of this region of the hippocampus, were not found to accompany new synapse formation. On a molecular level, both developmental and lesion data suggest that the expression of the synapsin I gene is tightly regulated in the central nervous system, and that considerable changes in synapsin I protein may occur in neurons without concomitant changes in the levels of its messenger RNA. Finally, our results suggest that the appearance of detectable levels of synapsin I protein in in developing and sprouting synapses coincides with the acquisition of function by those central synapses.

Synapsin I gene expression in the adult rat brain with comparative analysis of mRNA and protein in the hippocampus.

Synapsin I is the best characterized member of a family of neuron-specific phosphoproteins thought to be involved in the regulation of neurotransmitter release. In this report, we present the first extensive in situ hybridization study detailing the regional and cellular distribution of synapsin I mRNA in the adult rat brain. Both the regional distribution and relative levels of synapsin I mRNA established by in situ hybridization were confirmed by RNA blot analysis. Our data demonstrate the widespread yet regionally variable expression of synapsin I mRNA throughout the adult rat brain. The greatest abundance of synapsin I mRNA was found in the pyramidal neurons of the CA3 and CA4 fields of the hippocampus, and in the mitral and internal granular cell layers of the olfactory bulb. Other areas abundant in synapsin I mRNA were the layer II neurons of the piriform cortex and layer II and V neurons of the entorhinal cortex, the granule cell neurons of the dentate gyrus, the pyramidal neurons of hippocampal fields CA1 and CA2, and the cells of the parasubiculum. In general, the pattern of expression of synapsin I mRNA paralleled those encoding other synaptic terminal-specific proteins, such as synaptophysin, VAMP-2, and SNAP-25, with noteworthy exceptions. To determine specifically how synapsin I mRNA levels are related to levels of synapsin I protein, we examined in detail the local distribution patterns of both synapsin I mRNA and protein in the rat hippocampus. These data revealed differential levels of expression of synapsin I mRNA and protein within defined synaptic circuits of the rat hippocampus.

Alzheimer's disease and aging: effects on perforant pathway perikarya and synapses.

The hippocampal perforant pathway originates in the entorhinal cortex (ERC) and terminates in the outer molecular layer of the dentate gyrus (DG). To compare the effects of normal aging and Alzheimer's disease (AD) on the elements of the perforant pathway, we compared relative perikaryal numbers (determined by counting cell bodies and estimating volumes) in layer II of the ERC with synaptic quantities (estimated from immunoreactivity for the synaptic terminal protein synapsin I and DG volume) in the molecular layer of the DG. The brains of 5 young and 9 elderly cognitively normal individuals, and of 9 AD patients were studied. In normal aging we found a significant age-related decline in perikaryal numbers in the ERC without demonstrable synaptic loss in the DG. In AD there was marked and equivalent, (or proportional) reduction in both ERC perikaryal numbers and DG synapses. These data suggest that in normal aging remaining neurons may continue to support a full array of synapses, perhaps due to mechanisms such as axonal sprouting, synaptic enlargement, or synaptic ingrowth. In AD, however, the accelerated neuronal loss may overwhelm such compensatory mechanisms or alternatively, independent synaptic and perikaryal losses may occur.

Examination of X chromosome markers in Rett syndrome: exclusion mapping with a novel variation on multilocus linkage analysis.

Rett syndrome is a neurologic disorder characterized by early normal development followed by regression, acquired deceleration of head growth, autism, ataxia, and stereotypic hand movements. The exclusive occurrence of the syndrome in females and the occurrence of a few familial cases with inheritance through maternal lines suggest that this disorder is most likely secondary to a mutation on the X chromosome. To address this hypothesis and to identify candidate regions for the Rett syndrome gene locus, genotypic analysis was performed in two families with maternally related affected half-sisters by using 63 DNA markers from the X chromosome. Maternal and paternal X chromosomes from the affected sisters were separated in somatic cell hybrids and were examined for concordance/discordance of maternal alleles at the tested loci. Thirty-six markers were informative in at least one of the two families, and 25 markers were informative in both families. Twenty loci were excluded as candidates for the Rett syndrome gene, on the basis of discordance for maternal alleles in the half-sisters. Nineteen of the loci studied were chosen for multipoint linkage analysis because they have been previously genetically mapped using a large number of meioses from reference families. Using the exclusion criterion of a lod score less than -2, we were able to exclude the region between the Duchenne muscular dystrophy locus and the DXS456 locus. This region extends from Xp21.2 to Xq21-q23. The use of the multipoint linkage analysis approach outlined in this study should allow the exclusion of additional regions of the X chromosome as new markers are analyzed. This in turn will result in a defined region of the X chromosome that should be searched for candidate sequences for the Rett syndrome gene in both familial and sporadic cases.

A method for the direct measurement of mRNA in discrete regions of mammalian brain.

A rapid and nearly quantitative method for the direct analysis of steady-state mRNA levels in microgram quantities of frozen mammalian brain is described. Briefly, tissue punches 0.5-1.0 mm in diameter were sampled from 250-microns-thick cryostat sections of rat brain (approximately 50-200 micrograms tissue). The samples were homogenized in 50 microliters of a denaturing gel loading buffer and applied directly to a 2.2 M formaldehyde-agarose gel for electrophoresis and subsequent RNA blot analysis. The method is extremely rapid, results in excellent recovery of intact RNA, and allows the direct assay of mRNA levels in discrete subregions of the mammalian brain.

Positive- and negative-acting promoter sequences regulate cell type-specific expression of the rat synapsin I gene.

The phosphoprotein synapsin I is expressed exclusively in neuronal cells. We are interested in elucidating the promoter sequences involved in cell type-specific expression of the synapsin I gene. The PC12 cell line expresses the 3.4 kb and 4.5 kb synapsin I mRNAs and is used to analyze cell type-specific gene expression. A series of deletion fragments of the rat synapsin I gene promoter were fused to the promoterless reporter gene encoding bacterial chloramphenicol acetyltransferase (CAT) for transfection analysis in PC12 cells and in HeLa cells, which do not express the gene. A -349 bp to +110 bp rat synapsin I promoter fragment contains a positive regulator, shown to be 33-times more active in PC12 cells than HeLa cells. Transfection of reporter plasmids containing up to 4.4 kb of rat synapsin I gene promoter sequences exhibit significantly reduced CAT activity in PC12 cells. The reduction in CAT expression was attributed to a negative regulator located between -349 bp and -1341 bp in the rat synapsin I promoter. Our results suggest that both positive and negative-acting sequence elements regulate cell type-specific expression of the rat synapsin I gene.

A highly polymorphic dinucleotide repeat on the proximal short arm of the human X chromosome: linkage mapping of the synapsin I/A-raf-1 genes.

A compound (AC)n repeat located 1,000 bp downstream from the human synapsin I gene and within the last intron of the A-raf-1 gene has been identified. DNA data-base comparisons of the sequences surrounding the repeat indicate that the synapsin I gene and the A-raf-1 gene lie immediately adjacent to each other, in opposite orientation. PCR amplification of this synapsin I/A-raf-1 associated repeat by using total genomic DNA from members of the 40 reference pedigree families of the Centre d'Etude du Polymorphisme Humaine showed it to be highly polymorphic, with a PIC value of .84 and a minimum of eight alleles. Because the synapsin I gene has been mapped previously to the short arm of the human X chromosome at Xp11.2, linkage analysis was performed with markers on the proximal short arm of the X chromosome. The most likely gene order is DXS7SYN/ARAF1TIMPDXS255DXS146, with a relative probability of 5 x 10(8) as compared with the next most likely order. This highly informative repeat should serve as a valuable marker for disease loci mapped to the Xp11 region.

Synapsin I expression in the rat retina during postnatal development.

The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.

Synaptic loss in Alzheimer's disease and other dementias.

The extent and location of neuronal losses necessary or sufficient to produce dementia in patients with Alzheimer's Disease (AD) is unknown. To approach this question, we studied synaptic terminals in postmortem brain tissue utilizing immunohistochemical techniques. We used antibodies against two proteins found in synaptic terminals--synapsin I and synaptophysin--as synaptic markers in the hippocampal complexes of eight patients with autopsy-proven AD and eight nondemented control subjects. Quantitative microscopy measured the regional density of synaptic staining. All AD patients showed a striking decrease in synaptic staining in the outer half of the molecular layer of the dentate gyrus compared with control brains, where the density of synaptic terminals was uniform throughout. In an additional patient with progressive degenerative dementia but without plaques or tangles on neuropathologic examination, similar depletion of synaptic staining was seen in the dentate gyrus. Quantitative densitometric analyses confirmed the focal decrease in synaptic staining in the outer half of the molecular layer in demented patients. We also found a slight increase in synaptic staining in the inner half of this layer.

Multiple synapsin I messenger RNAs are differentially regulated during neuronal development.

Synapsin I is a neuron-specific protein consisting of two isoforms Ia and Ib. It is thought to play a role in the regulation of neurotransmitter release. In this study the structure and expression of two classes of synapsin I mRNA have been examined. The two mRNA classes have molecular sizes of 3.4 and 4.5 kb, respectively. Both classes translate into synapsin I polypeptides and display a high degree of base sequence homology. Utilizing an oligonucleotide-directed RNase H assay we have shown that both mRNA classes have a common start site of transcription and differ from one another toward their 3' ends. The expression of the two synapsin I mRNA classes is differentially regulated during the development of the rat brain and cerebellum. In the cerebellum the 4.5-kb transcript is expressed until postnatal day 7, after which it decreases to an undetectable level. The 3.4-kb mRNA is found throughout cerebellar development and in the adult. This suggests that the 3.4-kb mRNA class consists of messages which can encode both synapsin I polypeptides. Using quantitative Northern blot analysis a peak in the expression of this mRNA was observed at postnatal day 20. The maximum expression of the 3.4-kb class coincides with the period of synaptogenesis in the cerebellum. In addition to the developmental time course of synapsin mRNA expression a description of its spatial distribution throughout the cerebellum was performed using in situ hybridization histochemistry. From postnatal day 15 onwards, with a maximum at postnatal day 20, synapsin mRNA was localized in the internal granule cell layer of the cerebellum. On a cellular level, the granule cells, but not the neighboring Purkinje cells, express high levels of synapsin mRNA. These observations implicate developmentally coordinated differential RNA splicing in the regulation of neuron-specific gene expression and substantiate the correlation of synapsin gene expression with the period of synaptogenic differentiation of neurons.

Molecular analysis of synapsin I, a candidate gene for Rett syndrome.

The characteristics of Rett syndrome suggest that it is an X-linked neurodegenerative disorder. Laboratory investigations to date have not revealed any metabolic abnormalities in affected individuals. Synapsin I is a neuron-specific protein thought to play a fundamental role in neuronal function. In this report we summarize the circumstantial evidence suggesting that a defect in synapsin I gene structure or expression might be involved in Rett syndrome. This evidence includes analysis of structural and functional aspects of synapsin I primary structure, characterization of synapsin I messenger RNAs, location of the synapsin I gene on the human X chromosome and preliminary analysis of synapsin I gene structure in Rett individuals.

Determination and analysis of the primary structure of the nerve terminal specific phosphoprotein, synapsin I.

A rat brain cDNA clone containing an open reading frame encoding the neuron-specific protein synapsin I has been sequenced. The sequence predicts a protein of 691 amino acids with a mol. wt of 73 kd. This is in excellent agreement with the size of rat brain synapsin Ib measured by SDS--polyacrylamide gel electrophoresis. Inspection of the predicted primary structure has revealed the probable sites for synapsin I phosphorylation by the cAMP-dependent and Ca2+/calmodulin-dependent protein kinases. All of the biochemically observed intermediates of synapsin I digestion by collagenase can be verified by inspection of the sequence, and the collagenase-resistant fragment has been defined as the amino-terminal 439 amino acids of the molecule. Predictions of sequence secondary structure and hydrophobicity suggest that a central domain of approximately 270 amino acids may exist as a folded, globular core. The carboxyl-terminal domain of the protein (the region sensitive to collagenase digestion) contains sites for Ca2+/calmodulin-dependent protein kinase phosphorylation. These sites are flanked by three regions of repeating amino acid sequence that are proposed to be the synaptic vesicle-binding domain of synapsin I. This region also shares homology with the actin-binding proteins profilin and villin. The characteristics of the synapsin I sequence do not support extensive homology with the erythrocyte cytoskeletal protein 4.1.

Genes for synapsin I, a neuronal phosphoprotein, map to conserved regions of human and murine X chromosomes.

Synapsin I is a neuron-specific phosphoprotein associated with the membranes of small synaptic vesicles. Its function is not entirely clear, but evidence points to a possible role in the regulation of neurotransmitter release. Its biosynthesis is under developmental control. Assignment of the human synapsin I gene to the X chromosome at band Xp11 was accomplished by in situ hybridization, using a rat cDNA probe. Southern blot analysis of DNAs from a panel of human-Chinese hamster somatic cell hybrids with defined regions of the human X chromosome confirmed the in situ mapping data. The mouse synapsin I gene was assigned to the X chromosome, proximal to band XD, by Southern blot analysis of Chinese hamster-mouse somatic cell hybrids with normal or rearranged mouse X chromosomes. In situ chromosomal hybridization experiments localized the mouse synapsin I gene more precisely to bands XA1----A4. These results add to the comparative gene map of mammalian species and support certain hypotheses regarding the evolutionary relationship between human and mouse X chromosomes. We hypothesize that the synapsin I gene could be mutated in human X-linked disorders with primary neuronal degeneration, such as the Rett syndrome.

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.