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.

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.

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.

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 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.

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.

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.

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 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.

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.