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Biomedical subjects

M Jucker

Publications and source records attributed to M Jucker.

At least 37 records · Page 2Linked to original sources

Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology.

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid beta (A beta) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes A beta deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of A beta in the pathogenesis of the disease.

Alzheimer Disease↗

Laminins of the adult mammalian CNS; laminin-alpha2 (merosin M-) chain immunoreactivity is associated with neuronal processes.

Laminins are glycoproteins with three subunits, i.e. a longer alpha chain, a shorter beta chain and a shorter gamma chain. Well-characterized laminins are laminin-1 (EHS laminin; alpha1-beta1-gamma1), laminin-2 (merosin; alpha2-beta1-gamma1), laminin-3 (alpha1-beta2-gamma1) and laminin-4 (alpha2-beta2-gamma1). The present study shows that in the adult mammalian CNS (rat, rabbit, pig and monkey) alpha2 chain immunoreactivity is associated most evidently with neuronal fibers and punctate, potentially synaptic, structures of limbic brain regions. Third ventricle tanycytes and ensheathing cells of the olfactory nerve also express intense alpha2 chain immunoreactivity. Immunostaining for gamma1 chain is present throughout the central nervous system (CNS) in essentially all neuronal cell bodies and their most proximal processes. Immunoreactivity for all chains investigated (alpha1, alpha2, beta1, beta2 and gamma1) were present around blood vessels, especially evident in lightly fixed tissues. The finding that, other than blood vessels, neurons and other structures exhibited immunoreactivity for only one or two (and not three) chains, suggests that variant forms of laminin with yet undiscovered chains or other configurations than the heterotrimeric form are present in the CNS. The association of alpha2-like immunoreactivity with neuronal fibers and synaptic structures is of great interest in light of the known neurite-promoting and cell attachment activities of laminin-2.

Animals↗

Laminin-alpha2 chain-like antigens in CNS dendritic spines.

The laminin-alpha2 chain is a component of brain capillary basement membranes and appears also to be present in neurons of rat, rabbit, pig and non-human primate brain as evidenced by immunohistochemistry. In the present study, we have further characterized this very distinct neuronal laminin-alpha2 chain-like immunoreactivity in the hippocampus of various species. Immunoelectron microscopy with poly- and monoclonal antibodies to the laminin-alpha2 chain G-domain localized laminin-alpha2 chain immunoreactivity in adult rat and rabbit hippocampus to dendritic processes, primarily to dendritic spines. In the developing rat hippocampus, spine-associated laminin-alpha2 chain-like immunoreactivity first appeared at a time corresponding to that of active synaptogenesis. After an entorhinal cortex lesion in adult rats, the time course of denervation-induced loss and reactive reappearance of spines in the molecular layer of the dentate gyrus was correlated closely to the loss and reappearance of laminin-alpha2 chain immunoreactivity. Immunoblot analysis of normal adult rat, rabbit and pig brain revealed a protein similar in size to the reported 80-kDa laminin-alpha2 chain fragment of human placenta as well as 140/160-kDa proteins. These results suggest the presence of proteins with antigenic homology to the laminin-alpha2 chain and/or laminin-alpha2 isoforms in dendrites and dendritic spines in selected areas of the brain, predominately in the hippocampus and other limbic structures. Given the adhesion and neurite promoting functions of laminins, it is possible that neuronal laminin-alpha2 chain-like proteins play a role in synaptic function and plasticity in the CNS.

Animals↗

Murine models of brain aging and age-related neurodegenerative diseases.

In the past, structural changes in the brain with aging have been studied using a variety of animal models, with rats and nonhuman primates being the most popular. With the rapid evolution of mouse genetics, murine models have gained increased attention in the neurobiology of aging. The genetic contribution of age-related traits as well as specific mechanistic hypotheses underlying brain aging and age-related neurodegenerative diseases can now be assessed by using genetically-selected and genetically-manipulated mice. Against this background of increased demand for aging research in mouse models, relatively few studies have examined structural alterations with aging in the normal mouse brain, and the data available are almost exclusively restricted to the C57BL/6 strain. Moreover, many older studies have used quantitative techniques which today can be questioned regarding their accuracy. Here we review the state of knowledge about structural changes with aging in outbred, inbred, genetically-selected, and genetically-engineered murine models. Moreover, we suggest several new opportunities that are emerging to study brain aging and age-related neurodegenerative diseases using genetically-defined mouse models. By reviewing the literature, it has become clear to us that in light of the rapid progress in genetically-engineered and selected mouse models for brain aging and age-related neurodegenerative diseases, there is a great and urgent need to study and define morphological changes in the aging brain of normal inbred mice and to analyze the structural changes in genetically-engineered mice more carefully and completely than accomplished to date. Such investigations will broaden knowledge in the neurobiology of aging, particularly regarding the genetics of aging, and possibly identify the most useful murine models.

Aging↗

Cerebral lipid deposition in aged apolipoprotein-E-deficient mice.

To assess the influence of age and diet on cerebral pathology in mice lacking apolipoprotein E (apoE), four male apoE knockout mice (epsilon -/-), and five male wild-type (epsilon +/+) littermate controls were placed on a high-fat/high-cholesterol diet for 7 weeks beginning at 17 months of age. All four aged knockout mice developed xanthomatous lesions in the brain consisting mostly of crystalline cholesterol clefts, lipid globules, and foam cells. Smaller xanthomas were confined mainly to the choroid plexus and ventral fornix in the roof of the third ventricle, occasionally extending subpially along the choroidal fissure and into the adjacent parenchyma. More advanced xanthomas disrupted adjoining neural tissue in the fornix, hippocampus, and dorsal diencephalon; in one case, over 60% of one telencephalic hemisphere, including nearly the entire neocortex, was obliterated by the lesion. No xanthomas were observed in aged wild-type controls fed the high-fat/high-cholesterol diet. Brains from 42 additional animals, fed only conventional chow, were examined; 3 of 15 aged (15- to 23-month-old) apoE knockout mice developed small choroidal xanthomas. In contrast, no lesions were observed in five young (2- to 4-month-old) apoE knockout mice or in any wild-type controls between the ages of 2 and 23 months. Our findings indicate that disorders of lipid metabolism can induce significant pathological changes in the central nervous system of aged apoE knockout mice, particularly those on a high-fat/high-cholesterol diet. It may be fruitful to seek potential interactions between genetic factors and diet in modulating the risk of Alzheimer's disease and other neurodegenerative disorders in aged humans.

Aging↗

Comparative evaluation of synaptophysin-based methods for quantification of synapses.

Development, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the stereological optical dissector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the dissector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereology is the most useful method for estimating synaptic numbers in defined regions of the brain.

Animals↗

Laminin alpha 2 is a component of brain capillary basement membrane: reduced expression in dystrophic dy mice.

In the present study we demonstrate low level expression of the laminin alpha 2 chain in brain and localize the alpha 2 protein to the capillary basement membrane. While in peripheral basement membranes the laminin alpha 1 and alpha 2 chains have an almost mutually exclusive distribution, the present results suggest both alpha 1 and alpha 2 in the cerebral capillary basement membrane. Towards elucidating the function of alpha 2 in brain, we have performed ultrastructural analysis of the capillary basement membrane in dystrophic dy mice, which show a 70-90% and > 95% reduction of alpha 2 messenger RNA compared to heterozygous and wild-type mice, respectively, and show a nearly total absence of the alpha 2 protein by immunofluorescence. In contrast to the muscle and Schwann cell basement membrane, where alpha 2 deficiency causes structural basement membrane abnormalities, the present results show that the lack of the alpha 2 subunit in the cerebral capillary basement membrane is not detrimental to its structure. This observation might be explained by the fact that the cerebral capillary basement membrane expresses both alpha chains and therefore exhibits structural redundancy.

Animals↗

Similarities in the age-related hippocampal deposition of periodic acid-schiff-positive granules in the senescence-accelerated mouse P8 and C57BL/6 mouse strains.

With advancing age clusters of abnormal granules positive for periodic acid-Schiff appear in the hippocampus of C57BL/6 (B6) mice and the senescence-accelerated mouse (SAM) P8. The granules can also be visualized with a polyclonal antibody to a 110,000 mol. wt laminin-binding protein and stain specifically with a monoclonal antibody to heparan sulfate proteoglycan. The present study used light and electron-microscopic analysis to compare the staining and morphological properties of these granules in SAM P8 hippocampus with those in B6 hippocampus at different ages. The results of the light-microscopic analysis revealed that granules in SAM P8 and B6 had similar morphology, staining characteristics and distribution patterns, and appeared to have a close association with astrocytic process. The onset of granules in SAM P8 mice (at two to three months of age) was earlier than that observed in B6 mice (at four to six months of age), but the maximum incidence was similar in both strains. Electron-microscopic analysis revealed that the granules in SAM P8 and B6 mice also had a very similar ultrastructure. Granules in both strains were surrounded by a discontinuous membrane and contained mostly crystalline-like, degenerated material. The successive ultrastructural changes from the exterior to interior of the granules suggest that the degenerative process was initiated outside the granules and that degenerative structures migrate inward. Astrocytes and heparan sulfate proteoglycan are closely associated with beta-amyloid deposits in Alzheimer's disease. The presence of astrocyte-associated heparan sulfate proteoglycan-positive material in aged SAM P8 and B6 mice might model age-related alterations in glia function possibly involved in human cerebral amyloidogenesis.

Aging↗

Expression of the neural adhesion molecule L1 in the deafferented dentate gyrus.

Expression of the neural adhesion molecule L1 and its potential involvement in axonal sprouting were examined in the deafferented rat dentate gyrus. We focused on the dentate gyrus because of its well-defined cytoarchitecture and well-characterized neuronal degeneration and sprouting response following entorhinal cortex lesions. In the molecular layer of the dentate gyrus, a trilaminar staining pattern was observed, with the middle molecular layer exhibiting slightly denser immunolabeling compared to both inner and outer molecular layers. Two to 12 days after a unilateral entorhinal cortex lesion, a progressive loss of L1 immunolabeling was noted in the ipsilateral middle and outer molecular layers, followed by a substantial reappearance of immunostaining 65 days after lesion incidence. The width of the immunostained ipsilateral inner molecular layer revealed a progressive widening and by postlesion day 65 occupied about 50% of the total width of the molecular layer. Immunoelectron microscopy localized L1 to the surface of unmyelinated axons in both normal and deafferented dentate gyrus. In situ hybridization revealed L1 messenger RNA confined to neurons throughout the hippocampal formation, but did not indicate changes in L1 messenger RNA levels in the hippocampus, dentate gyrus, entorhinal cortex or basal forebrain in response to unilateral entorhinal cortex lesions. Changes in L1 immunolabeling in the deafferented dentate gyrus corresponded in a spatial and temporal manner to changes of the synaptic marker synaptophysin and axonal marker phosphorylated tau. Results of the present study are most consistent with the view that L1 is expressed on reinnervating fibers after they make synaptic contacts with other structures. Thus, L1 appears to be involved in the maturation and stabilization of reinnervating fibers and consequently may play an important role in the repair process of the lesioned adult CNS.

Animals↗

Laminin stimulates expression of two mitochondrial proteins during neurite outgrowth.

Differential hybridization was utilized with mRNA from NG108-15 cells cultured on either tissue culture plastic or laminin for 4 hr to identify genes whose mRNA was increased by laminin, a potent stimulator of neurite outgrowth. Two of the 16 laminin-induced clones, cytochrome b and chargerin II, are mitochondrial proteins. Northern blotting confirmed that laminin increased the mRNA levels of cytochrome b and chargerin II several fold. Antibody to chargerin II stained both processes and cell bodies of the cerebellar Purkinje cells and localized in the mitochondria of NG108-15 cells, which also showed increased protein levels in the presence of laminin. In addition, higher levels of chargerin II protein were detected in the newborn brain compared to the adult. However, inhibitors of mitochondrial protein synthesis did not affect laminin-mediated neurite outgrowth. These data suggest that the increased synthesis of mitochondrial enzymes observed with laminin treatment may not be necessary for the formation of neurites.

Animals↗

The effect of continuous intraventricular infusion of L1 and NCAM antibodies on spatial learning in rats.

Recent studies suggest a role of the neural cell adhesion molecules L1 and NCAM in mechanisms of memory storage. In the present study we analyzed the effect of continuous intraventricular infusion of polyclonal antibodies directed against L1 (antiL1) or NCAM (antiNCAM) on the performance of male Wistar rats during the acquisition and retention of a spatial learning task (Morris water-maze). In this task animals have to learn the spatial position of a hidden escape platform in a water tank to escape onto it. During acquisition of the task animals with continuous infusion of antiNCAM - but not those infused with antiL1 - showed day-dependent attenuated learning in comparison to controls (P = 0.001). Control animals were either injected with vehicle (PBS) or with polyclonal antibodies raised against liver cell membrane. When the escape platform was removed during the retention test (transfer test), the performance of animals continuously infused with antiL1 as well as those continuously infused with antiNCAM showed an impaired search pattern when compared with the performance of control animals (P = 0.001 and 0.04, respectively). Whereas control animals spent up to 46% of their time searching for the platform in the correct quadrant, the time antiL1- and antiNCAM-infused animals spent in this quadrant was closer to chance level (30.5% and 36.5%), respectively). The present data provide additional support for an involvement of the two adhesion molecules L1 and NCAM in synaptic plasticity underlying memory storage.

Animals↗

Dystroglycan in the cerebellum is a laminin alpha 2-chain binding protein at the glial-vascular interface and is expressed in Purkinje cells.

Dystroglycan is a core component of the dystrophin receptor complex in skeletal muscle which links the extracellular matrix to the muscle cytoskeleton. Dystrophin, dystrophin-related protein (DRP, utrophin) and dystroglycan are present not only in muscles but also in the brain. Dystrophin is expressed in certain neuronal populations while DRP is associated with perivascular astrocytes. To gain insights into the function and molecular interactions of dystroglycan in the brain, we examined the localization of alpha- and beta-dystroglycan at the cellular and subcellular levels in the rat cerebellum. In blood vessels, we find alpha-dystroglycan associated with the laminin alpha 2-chain-rich parenchymal vascular basement membrane and beta-dystroglycan associated with the endfeet of perivascular astrocytes. We also show that alpha-dystroglycan purified from the brain binds alpha 2-chain-containing laminin-2. These observations suggest a dystroglycan-mediated linkage between DRP in perivascular astrocytic endfeet and laminin-2 in the parenchymal basement membrane similar to that described in skeletal muscle. This linkage of the astrocytic endfeet to the vascular basement membrane is likely to be important for blood vessel formation and stabilization and for maintaining the integrity of the blood-brain barrier. In addition to blood vessel labelling, we show that alpha-dystroglycan in the rat cerebellum is associated with the surface of Purkinje cell bodies, dendrites and dendritic spines. Dystrophin has previously been localized to the inner surface of the plasma membrane of Purkinje cells and is enriched at postsynaptic sites. Thus, the present results also support the hypothesis that dystrophin interacts with dystroglycan in cerebellar Purkinje neurons.

Animals↗

A 110-kD nuclear shuttling protein, nucleolin, binds to the neurite-promoting IKVAV site of laminin-1.

The basement membrane protein laminin and the IKVAV-containing sequence from the laminin alpha 1 chain have been found to promote the differentiation of primary neurons and a variety of neural cell lines. We previously reported that a 110-kd IKVAV-binding protein (LBP110) isolated from brain appears to be a member of the beta-amyloid precursor protein (APP) family by immunologic and functional studies, which showed that LBP110/APP is also important in neurite outgrowth (Kibbey et al.: Proc Natl Acad Sci USA 90:10150-10153, 1993). In the preparation of this binding protein, a contaminating IKVAV-binding protein of identical molecular weight, nucleolin, was also identified. Here we have studied the relationship between these binding proteins. We find that nucleolin binds specifically to the IKVAV sequence independently of LBP110/ApP. We have also demonstrated significant levels of nucleolin in mature brain and in differentiating neural cells, suggesting that nucleolin functions not only in cell proliferation and in ribosome biogenesis as was previously reported, but also in the differentiation and maintenance of neural tissue. Our identification of cytoplasmic and cell-surface nucleolin, an IKVAV-binding protein, suggests that this protein may function in signalling by extra-cellular matrix.

Amino Acid Sequence↗

Transient upregulation of NCAM mRNA in astrocytes in response to entorhinal cortex lesions and ischemia.

Axonal sprouting and synaptic reorganization play an important role in the adaptation of the CNS to injury. However, the molecular mechanisms underlying this neuronal plasticity are poorly understood. In the present study we used in situ hybridization to examine the expression of NCAM mRNA in normal hippocampus, and in response to entorhinal cortex (EC) lesions and transient global ischemia. Both neurons and astrocytes were labeled by digoxygenin-tagged cRNA probes which recognize all three major NCAM isoforms of the adult CNS. In contrast, NCAM180-specific probes labeled only neurons in the hippocampus. After unilateral EC lesion, a transient and anatomically restricted upregulation of NCAM120/140 mRNA in reactive astrocytes in the denervated molecular layer of the dentate gyrus was observed. This increase was only present 2-4 days after the lesion whereas the GFAP mRNA increase was present up to 30 days postlesion. Following global ischemia a similar, transient increase of NCAM120/140 mRNA labeling of reactive astrocytes was observed; this increase was anatomically restricted to CA1, where neuronal loss occurred. Results suggest that the transient upregulation of NCAM120/140 mRNA in reactive astrocytes shortly after injury might be an important molecular mechanism in the cascade of events underlying neuronal plasticity in the adult CNS.

Animals↗

Age-related fibrillar material in mouse brain. Assessing its potential as a biomarker of aging and as a model of human neurodegenerative disease.

We have described the age-related deposition of fibrillar material in brains of B6 mice and SAM. Since in other inbred strains similar deposits were absent or occurred only occasionally and only in aged individuals, a genetic predisposition of B6 mice and SAM to accumulate the fibrillar material is suggested. The deposits are mostly associated with astrocytic processes and have been referred to as astrocytic inclusions. HSPG- and laminin-like molecules have been identified as components of the fibrillar material. The deposits have similarities with CA in humans, but they also show some important differences; thus there is presently insufficient evidence to consider the deposits the murine equivalent of CA. Although the physiological significance of the fibrillar material is not yet clear, the awareness of the deposits appears pertinent because they might contribute to various aspects of CNS function of susceptible strains of mice, and therefore could lead to possible misinterpretations of the results of studies employing these strains. Future directions of our research will determine the potential of the murine deposits to model aspects of human neuropathology, in particular, whether the deposits may mimic the deposition of ECM molecules as an early-event in the pathogenesis of amyloid plaque formation.

Aging↗

Age-related deposition of glia-associated fibrillar material in brains of C57BL/6 mice.

With advancing age, clusters of unusual granules appear in the brains of C57BL/6 (B6) mice. At the light, confocal laser and electron microscopic levels, the granules represent aggregations of fibrillar material often associated with astrocytes. The fibrillar material is largely free of normal organelles and has been located within astrocytic somata and processes, although in many cases the material is found in the neuropil and is surrounded by a discontinuous membrane. The deposits occur predominantly in hippocampus, but also in piriform cortex, cerebellum and less frequently in some other brain regions. They become evident about six months of age and increase markedly in both number and size thereafter. Incidence of the deposits varies greatly among inbred mouse strains. At six to 12 months of age, granules are abundant in male and female B6, and are absent in BALB/c, CBA, DBA/2 and A mice. In hybrid strains with a B6 background the deposits are also present and thus appear to manifest dominant genetic heritability. Similar granular structures have been described in adult brains of the senescence accelerated mouse and have been noted, albeit very rarely, in aged mice from other strains. While immunostaining of the granules with several polyclonal antisera was found by preabsorption with antigens to be non-specific, immunolabeling with monoclonal antibodies to heparan sulfate proteoglycan core protein and to laminin suggest these or related molecules as components of the fibrillar material. The presence of glycosaminoglycans is supported by staining with periodic acid-Schiff and Gomori's methenamine silver methods. The functional significance of the murine deposits is not yet clear. The deposits do not represent senile plaques with beta-amyloid deposition, but they might mimic the deposition of extracellular matrix molecules that is hypothesized to be a precursor condition for plaque formation and cerebral amyloidosis. Furthermore, the genetic differences in the incidence of the fibrillar deposits has potential to model aspects of familial neurodegenerative diseases.

Aging↗

beta-Amyloid precursor protein binds to the neurite-promoting IKVAV site of laminin.

We previously characterized a 110-kDa membrane-associated laminin-binding protein (LBP110) from brain which binds the laminin A chain -Ile-Lys-Val-Ala-Val-(IKVAV) site and increases in injury. Here we demonstrate that antisera directed against different epitopes of beta-amyloid precursor protein (APP) recognize LBP110 and that APP is recognized by LBP110 antiserum. APP specifically binds IKVAV and not another biologically active laminin-derived peptide containing the amino acid sequence -Tyr-Ile-Gly-Ser-Arg-. PC-12 cells transfected with antisense APP RNA produce less APP and LBP110, and they form fewer processes when cultured on either laminin or the IKVAV peptide. Thus, LBP110 is a member of the APP family and a function for APP in neurite outgrowth is now defined.

Amino Acid Sequence↗