Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) isoforms

The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (alpha-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1 A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca2+- and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca2+ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or alpha-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

Are mechanisms of exocytosis neurotransmitter specific?

In their review, Langley and Grant (1997) investigate the question whether mechanisms of exocytosis are neurotransmitter specific. There is now much evidence that the mechanisms governing the exocytosis of the two principal storage organelles--granules (large dense core vesicles) and electron-lucent vesicles--differ. But much less is known concerning potential differences in the release mechanisms of electron-lucent vesicles that store different types of fast neurotransmitters or of granules in different types of neurons. It is an open question whether there is a unifying control mechanism for the exocytosis of, for example, a peptide-containing granule of a glutamatergic neuron, a chromaffin granule, a noradrenergic granule or a granule from a neurosecretory neuron in the pituitary. The small electron-lucent synaptic vesicles of various kind apparently share common molecular components of regulated release. They carry the calcium sensor synaptotagmin, small GTP-binding proteins of the rab3 group or the v-SNARE synaptobrevin. Nevertheless, there may be differences in the regulatory mechanisms. This concerns the type of calcium channel involved or the absence of some of the presynaptic molecules such as rab3a, synapsin I or the t-SNAREs SNAP-25 or syntaxin from distinct types of neurons or sensory cells.     

Exocytotic proteins in enterochromaffin-like (ECL) cells of the rat stomach

Proteins participating in vesicular docking and fusion have been identified in the nervous system. Such proteins appear to be important for the molecular regulation of exocytosis also in non-neuronal cells. The enterochromaffin-like (ECL) cells of the gastric acid-secreting (oxyntic) mucosa secrete histamine and chromogranin A-derived peptides, such as pancreastatin. Using immunohistochemistry, we have examined whether the ECL cells of the rat stomach, identified with antibodies to histidine decarboxylase (HDC, the histamine-forming enzyme), express the same exocytotic proteins as neurons. The ECL cells displayed immunoreactivity for synaptophysin, synaptotagmin III, vesicle-associated membrane protein-2 (VAMP-2), cysteine string protein (CSP), vesicular monoamine transporter-2 (VMAT-2), synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin, and Munc-18, but not for synaptotagmin I/II and VAMP-1. Synaptophysin and VMAT-2 could be detected not only in the ECL cells, but also in a population of HDC-negative cells. The demonstration of synaptotagmin III in only a limited number of ECL cells suggests the existence of a subpopulation of ECL cells. The results show that several exocytotic proteins, previously identified in neurons, are present in rat stomach ECL cells. Hence, proteins engaged in vesicular docking and in the fusion of granule/vesicle membrane with plasma membrane seem to exist in both neurons and endocrine cells.     

Differential expression of rat brain synaptic proteins in development and aging

We have previously reported the differential involvement of synaptic proteins in Alzheimer's disease (AD). As AD is an aging-associated disease, in the present study we examined the developmental and aging-related changes in synaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), syntaxin 1/HPC-1 and drebrin in the rat brain. Immunoblot analyses of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that the protein level of synaptophysin and synaptobrevin increased after birth, being highest at 24 weeks, and then decreased with aging. Synaptotagmin was detected at E19, with levels increasing after birth to 96 weeks. SNAP-25 levels were highest at 4 weeks, and then decreased with aging. Syntaxin 1/HPC-1 levels were high at E19 and 1 week, decreasing rapidly from 2 weeks onwards, and drebrin levels were highest at E19 and 1 week, and decreased during aging. The present results suggest that the expression of each synaptic protein is differentially regulated in development and aging.     

Effect of chronic haloperidol treatment on synaptic protein mRNAs in the rat brain

Chronic haloperidol treatment caused significant decreases in the levels of synaptotagmin I and IV, synaptobrevin II, syntaxin 1A and Rab 3A mRNAs in the nucleus accumbens but not in the prefrontal cortex medial field, striatum, substantia nigra and ventral tegmental area. No significant changes in SNAP 25 and synaptophysin mRNA levels were observed in any brain region examined. The reduced expression of synaptic proteins may be related to haloperidol-induced depolarization block of mesolimbic dopamine neurons.     

Characterization of SNARE protein expression in beta cell lines and pancreatic islets

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.     

The proteins synaptotagmin and syntaxin are not general targets of Lambert-Eaton myasthenic syndrome autoantibody

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular disease in which impairment of Ca2+ entry into the nerve ending and consequent impaired release of acetylcholine (ACh) results in muscle weakness. The identity of the primary antigenic target molecule(s) of the autoantibodies is uncertain. Electrophysiological studies and 45Ca2+ uptake studies implicate a direct effect on the Ca2+ channel complex at the motor nerve terminal. Some recent studies, however, suggest a more indirect interference caused by binding of autoantibodies to synaptotagmin or syntaxin, molecules presumed to be involved in docking and/or coupling the synaptic vesicles to the Ca2+ channels in the active zone for vesicle exocytosis and transmitter release. Western blot analyses of rat and human brain membrane proteins and pure recombinant synaptotagmin and syntaxin were used to examine directly the targets of LEMS autoantibodies and determine specifically whether or not synaptotagmin and/or syntaxin were general targets in LEMS. IgG from 14 patients with LEMS was used to probe western blots of gels containing synaptotagmin, syntaxin, rat synaptosomal proteins, and human brain membrane proteins. Several similar immunoreactive bands were observed using both rat and human brain membranes. These include high-molecular-weight protein bands whose size would be consistent with being components of Ca2+ channels. No reactive component was observed against either syntaxin or synaptotagmin in IgG of the 14 LEMS patients. However, both human and rat brain membranes contain proteins recognized by antibodies directed against synaptotagmin or syntaxin, indicating their immunologic relatedness and evolutionary conservation. These results suggest that large-molecular-weight proteins consistent with being Ca2+ channel subunits rather than syntaxin and synaptotagmin are general targets of LEMS autoantibodies.     

Interaction of cysteine string proteins with the alpha1A subunit of the P/Q-type calcium channel

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.     

Immortalized gonadotropin-releasing hormone neurons secrete gamma-aminobutyric acid-evidence for an autocrine regulation

The immortalized hypothalamic neuronal cell lines GT1-1 and GT1-7 represent unique model systems to investigate the physiological control of gonadotropin-releasing hormone (GnRH) secretion. Using immunofluorescence microscopy, key proteins of regulated exocytosis, e.g. synaptotagmin, synaptobrevin and SNAP-25 (synaptosomal associated protein of 25 kDa) were found in GT1 neurons. In addition, GT1 neurons contained synaptophysin, a marker protein for small synaptic vesicles (SSVs) which are responsible for the storage of neurotransmitters such as gamma-aminobutyric acid (GABA). Upon subcellular fractionation, a lighter vesicle population characterized by synaptophysin separated from a denser vesicle population containing GnRH. Both vesicle populations contained synaptobrevin and synaptotagmin. Besides GnRH, GT1 neurons expressed glutamic acid decarboxylase at the mRNA-level and synthesized GABA. More importantly, GT1 neurons took up and stored 3H-GABA. The stored GABA was released after stimulation with increasing K+ concentrations and by alpha-latrotoxin. Reducing the extracellular Ca2+-concentration abolished stimulated secretion, indicating that GABA was released by regulated exocytosis. Hormone secretion from GT1 neurons is controlled by GABA via GABA(A) and GABA(B) receptors reflecting the situation in vivo. Our data provide the first evidence that GT1 neurons possess a second regulated secretory pathway sustained by SSVs storing and releasing GABA. The released GABA influences GnRH secretion by an auto- or paracrine loop.     

Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.     

Increased association of synaptosome-associated protein of 25 kDa with syntaxin and vesicle-associated membrane protein following acrosomal exocytosis of sea urchin sperm

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated integral membrane protein expressed almost exclusively in neuronal and neuroendocrine tissues. This protein forms a ternary complex with vesicle-associated membrane protein (VAMP) and syntaxin, which is thought to regulate the fusion of plasma and vesicle membranes during exocytosis. We report the identification of SNAP-25 expressed in sea urchin sperm. Sea urchin SNAP-25 shares greater identity with mammalian SNAP-25 than with mammalian SNAP-23, a ubiquitously expressed homologue believed to regulate membrane fusion in non-neuronal tissues. Sea urchin sperm contain a single exocytotic vesicle, the acrosomal vesicle, whose contents are exposed during the acrosome reaction. Fusion of the plasma membrane with the acrosomal vesicle membrane at multiple points (vesiculation) results in the release of SNAP-25 with the shed acrosome reaction vesicles. A complex containing SNAP-25, syntaxin, and VAMP is present in sperm, as detected by affinity chromatography and immunoprecipitation. Although this complex is present prior to the acrosome reaction, the amount of complex increases over 4-fold following acrosomal exocytosis. These findings support the involvement of SNAP-25 in the invertebrate sperm acrosome reaction, possibly through increased association with VAMP and syntaxin driving the fusion of plasma and acrosomal membranes.     

Proteolysis of SNAP-25 by types E and A botulinal neurotoxins

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex.     

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion

The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.     

Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation

SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors alpha-, beta-, and gamma-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant alpha-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in alpha-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.     

The soluble N-ethylmaleimide-sensitive factor attached protein receptor complex in growth cones: molecular aspects of the axon terminal development

Soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE) mechanisms are thought to be involved in two important processes in axonal growth cones: (1) membrane expansion for axonal growth and (2) vesicular membrane fusion for mature synaptic transmission. We investigated the localization and interactions among the proteins involved in SNARE complex formation in isolated growth cone particles (GCP) from forebrain. We demonstrated that the SNARE complex is present in GCPs morphologically without synaptic vesicles (SVs) and associated with growth cone vesicles. However, the apparently SV-free GCP was lacking in the regulatory mechanisms inhibiting SNARE complex formation proposed in SV fusion, i.e., the association of synaptotagmin with the SNARE complex, and vesicle-associated membrane protein (VAMP)-synaptophysin complex formation. The core components of the SNARE complex (syntaxin, SNAP-25, and VAMP) accumulated for several days before postnatal day 7, when SVs first appeared, and preceded the accumulation of marker proteins such as synaptophysin, SV2, and V-ATPase. Our present results suggest that the SNARE mechanism for vesicular transmitter release is not fully functional in growth cones before the appearance of SVs, but the SNARE mechanism is working for membrane expansion in growth cones, which supports our recent report. We concluded that the regulation of the SNARE complex in growth cones is different from that in mature presynaptic terminals and that this switching may be one of the key steps in development from the growth cone to the presynaptic terminal.     

Axonal cytoskeletal pathology in aged and diabetic human sympathetic autonomic ganglia

Prevertebral sympathetic ganglia develop markedly enlarged argyrophilic neurites as a function of age, gender and diabetes. Immunolocalization studies demonstrate their preferential labeling with antisera to highly phosphorylated 200 kDa neurofilament (NF-H) epitopes, NPY, peripherin and synapsin I, but not to hypophosphorylated NF-M and NF-H or MAP-2. The immunophenotype of dystrophic neurites in conjunction with the results of histochemical and ultrastructural studies are consistent with the terminal axonal and/or synaptic origin of neuritic dystrophy in the sympathetic ganglia of aged and diabetic human subjects.     

Characterization of presynaptic proteins involved in synaptic vesicle exocytosis in the nervous system of Torpedo marmorata

Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a key role in the exocytotic machinery of synaptic vesicles. Their presence, distribution and interactions are reported in central and peripheral nervous systems of the electric fish Torpedo marmorata. These three proteins form a protein complex in all the nervous system regions tested, including the electric lobe and the electric organ which is innervated by pure cholinergic nerve terminals. Immunoblot analysis revealed a double protein pattern of SNAP-25 in the anterior brain and cerebellum, although a single protein band corresponding to SNAP-25 was observed in the electromotor system. Moreover, SNAP-25 showed a differential distribution in the electromotor system. It was present along nerve fibres and terminals that innervated the electric organ but it was not detected in nerve terminals at the electric lobe. Immunoisolation experiments using anti-synaptobrevin antibodies showed a tissue-specific co-existence of SNAP-25 and syntaxin with synaptobrevin in the immunoisolated organelles. In conclusion, the molecular components of the exocytotic machinery are shown to be conserved in Torpedo, although some differences mainly on SNAP-25, suggest a potential diversity in the regulation of neurosecretion.     

SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.     

Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve

Synaptophysin and synapsin, closely correlated on synaptic vesicles in terminals, may show a differential distribution at synapse formation and maturation. In order to disclose the fine structural details of these differences, synapsin and synaptophysin distribution was studied by immunocytochemistry on ligated bovine splenic axons in vitro and compared with terminals in the vas deferens. In the synaptic differentiations taking place proximally synapsin could only be detected on the accumulating elements of the axonal reticulum. Large dense granules and clusters of small synaptic vesicles were negative. Synaptophysin was restricted to these clusters. In the vas deferens, co-localization of synapsin and synaptophysin could be seen on small vesicles. From their formation small synaptic vesicles carry synaptophysin. Synapsin may be involved in the dynamic membrane changes taking place at the ligation. At a functional terminal, synapsin shifts to small synaptic vesicles.     

Comparison of neurodegenerative pathology in transgenic mice overexpressing V717F beta-amyloid precursor protein and Alzheimer's disease

Overexpression of mutated human amyloid precursor protein (hAPP717V-->F) under control of platelet-derived growth factor promoter (PDAPP minigene) in transgenic (tg) mice results in neurodegenerative changes similar to Alzheimer's disease (AD). To clarify the pathology of these mice, we studied images derived from laser scanning confocal and electron microscopy and performed comparisons between PDAPP tg mice and AD. Similar to AD, neuritic plaques in PDAPP tg mouse contained a dense amyloid core surrounded by anti-hAPP- and antineurofilament-immunoreactive dystrophic neurites and astroglial cells. Neurons were found in close proximity to plaques in PDAPP tg mice and, to a lesser extent, in AD. In PDAPP tg mice, and occasionally in AD, neuronal processes contained fine intracellular amyloid fibrils in close proximity to the rough endoplasmic reticulum, coated vesicles, and electron-dense material. Extracellular amyloid fibrils (9-11 nm in diameter) were abundant in PDAPP tg and were strikingly similar to those observed in AD. Dystrophic neurites in plaques of PDAPP tg mouse and AD formed synapses and contained many dense multilaminar bodies and neurofilaments (10 nm). Apoptotic-like figures were present in the tg mice. No paired helical filaments have yet been observed in the heterozygote PDAPP tg mice. In summary, this study shows that PDAPP tg mice develop massive neuritic plaque formation and neuronal degeneration similar to AD. These findings show that overproduction of hAPP717V-->F in tg mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular degenerative changes associated with AD.