Biomedical subjects

K L Moya

Publications and source records attributed to K L Moya.

At least 19 recordsLinked to original sources

PubMed2005

siRNA targeted against amyloid precursor protein impairs synaptic activity in vivo.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD) pathogenesis through its cleavage leading to the accumulation of the peptide betaA4. Diffusible oligomeric assemblies of amyloid beta peptide are thought to induce synaptic dysfunction, an early change in AD. We tested the hypothesis that a reduction in presynaptic APP could itself lead to a decrease in synaptic efficacy in vivo. Twenty-four hours after intraocular injection, siRNA targeted against APP accumulated in retinal cells and the APP in retinal terminals in the superior colliculus was significantly reduced. Surprisingly, the amyloid precursor-like protein 2 (APLP2) was reduced as well. Functional imaging experiments in rats during visual stimulation showed that knockdown of presynaptic APP/APLP2 significantly reduced the stimulation-induced glucose utilization in the superior colliculus. Our results suggest that perturbations in the amount of APP/APLP2 axonally transported to, and/or in their turnover in the nerve terminal alter synaptic function and could be a pathogenic mechanism in AD.

Amyloid beta-Protein Precursor↗

PubMed2001

High expression and anterograde axonal transport of aminoterminal sonic hedgehog in the adult hamster brain.

Sonic hedgehog (SHH) is considered to play an important role in tissue induction and patterning during development, particularly in determining neuronal cell fate in the ventral neural tube and in the embryonic forebrain. SHH precursor is autoproteolytically cleaved to an aminoterminal fragment (SHHN) which retains all known SHH biological activities. Here, we demonstrate the expression of a 22-kDa SHHN immunoreactive peptide in developing and adult hamster brain regions using a rabbit antiserum directed against a mouse SHHN fragment. Interestingly, SHHN was developmentally regulated with the highest expression observed in the adult brain, was resistant to Triton X-100 solubilization at 4 degrees C and partitioned with the raft component ganglioside GM1 during density gradient centrifugation. In rat brain, Shh transcripts were identified by double in situ hybridization in GABAergic neurons located in various basal forebrain nuclei including globus pallidus, ventral pallidum, medial septum-diagonal band complex, magnocellular preoptic nucleus and in cerebellar Purkinje cells as well as in motoneurons of several cranial nerve nuclei and of the spinal cord. We show that radiolabelled SHHN peptides are synthesized in the adult hamster retina and are transported axonally along the optic nerve to the superior colliculus in vivo. Our data indicate that SHHN is associated with cholesterol rich raft-like microdomains and anterogradely transported in the adult brain, and suggest that the roles of this extracellular protein are more diverse than originally thought.

Age Factors↗

PubMed2000

Early postnatal expression of L1 by retinal fibers in the optic tract and synaptic targets of the Syrian hamster.

Previous immunohistochemical studies in mouse, rat, and chick have reported that the expression of the glycoprotein and cell adhesion molecule L1, a member of the immunoglobulin superfamily, shows regulation during development of retina and optic nerve. To extend our understanding of the role of L1 in developing neural circuitry, we have examined L1 expression in the optic tract and thalamic and midbrain synaptic targets of retinal fibers in the early postnatal Syrian hamster, a well-characterized developmental model of the primary visual projection. Metabolic labeling studies reveal that a synaptically targeted, sulfated, and glycosylated form of L1 undergoes rapid axonal transport from the retina. Retinofugal transport of L1 decreases commensurate with the decline in immunoreactivity of retinal fibers in the visual pathway. Retinal ganglion cell axons show intense L1 immunoreactivity as they navigate in highly fasciculated bundles in the optic tract overlying the lateral geniculate body and in the superior colliculus. We found no evidence of L1 immunoreactivity on retinal axon collaterals as they defasciculate from the optic tract and branch into target neuropils. L1 immunoreactivity wanes in optic tract as axon terminal arbors are elaborated in the lateral geniculate body and superior colliculus and as myelination in the visual pathway commences. This pattern of L1 expression suggests that, in the early postnatal period, L1 may support fasciculation of retinal fibers, maintaining them within the optic tract, and that subsequent down-regulation of L1 may facilitate their terminal arborization and myelination.

Age Factors↗

PubMed2000

Immunolocalization of the cellular prion protein in normal brain.

We examined the localization of PrP(c) in normal brain using free-floating section immunohistochemistry and monclonal antibody 3F4. In the mature hamster and baboon brain, PrP(c) is localized to the neuropil with a synaptic distribution and the PrP(c) immunoreactivity is denser in regions known for ongoing plasticity. Cell bodies and major fiber tracts have little or no PrP(c) immunoreactivity. At the electron microscopic level, PrP(c) immunoreactivity decorates synaptic profiles, both pre- and postsynaptically. Results obtained with two additional antibodies, 3B5 and Pri-304, showed similar patterns of PrP(c) bands on Western blots, although Pri-304 was less sensitive. On sections through the adult hamster hippocampus, 3B5 and Pri-304 both stained the synaptic neuropil while cell bodies in the pyramidal and dentate granule cell layers were not immunoreactive. Pri-304 differentiated between synaptic layers in the hippocampus and closely resembled the pattern of staining obtained with 3F4. Preliminary results of developing brain showed that PrP(c) is initially localized along fiber tracts in the neonate brain. These results show that PrP(c) has a synaptic distribution in the adult brain and suggest that there are important changes in its distribution during brain development. These results also characterize two additional reagents for studies of PrP(c) localization.

Adult↗

PubMed2000

Inhibition of N-glycan processing alters axonal transport of synaptic glycoproteins in vivo.

Synaptic glycoproteins are synthesized and glycosylated in the neuronal cell body, and conveyed to terminals by fast axonal transport. We used the alpha-mannosidase inhibitor, 2-deoxymannojirimycin (dMan), to investigate the effects of disrupting N-glycan processing on the axonal trafficking of proteins in vivo. dMan significantly reduced rapid axonal transport in retinal ganglion cells to about 34% of control values 4h after metabolic labeling; at 8 h post-labeling the inhibition was reversed. 2-D gel analysis showed that dMan completely inhibited the arrival of radiolabeled L1 and NCAM at axon terminals, and resulted in the appearance of two novel proteins of 230 kDa and 155 kDa. Our results show that disruption of the N-glycosylation pathway has an immediate inhibitory effect on total axonal transport and longer lasting effects on the trafficking of specific glycoproteins to axon terminals in vivo.

1-Deoxynojirimycin↗

PubMed1999

A novel cellular prion protein isoform present in rapid anterograde axonal transport.

We studied the axonal transport of PrP(C) in hamster retinal and sciatic nerve axons. Our results show that a novel 38kDa form is the predominant form in rapid anterograde axonal transport while the 36kDa and 33kDa PrP(C) forms, abundant in nerve and brain, appear to be either stationary or slowly transported. We did not detect any significant retrograde transport of PrP(C). These results show that 38kDa PrP(C) is the form exported from the cell body to the axonal compartment where it may represent the precursor to the more abundant PrP(C) forms after its modification in nerve fibres or terminals.

Animals↗

PubMed1998

Post-translational processing and turnover kinetics of presynaptically targeted amyloid precursor superfamily proteins in the central nervous system.

The amyloid precursor superfamily is composed of three highly conserved transmembrane glycoproteins, the amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1, APLP2), whose functions are unknown. Proteolytic cleavage of APP yields the betaA4 peptide, the major component of cerebral amyloid in Alzheimer's disease. Here we show that five post-translationally modified, full-length species of APP and APLP2 (but not APLP1) arrive at the mature presynaptic terminal in the fastest wave of axonal transport and are subsequently rapidly cleared (mean half-life of 3.5 h). Rapid turnover of presynaptic APP and APLP2 occurs independently of visual activity. Turnover of the most rapidly arriving APP species was accompanied by a delayed accumulation of a 120-kDa, APP fragment lacking the C terminus, consistent with presynaptic APP turnover via constitutive proteolysis. Turnover of APLP2 was not accompanied by detectable APLP2 fragment peptides, suggesting either that APLP2 either is more rapidly degraded than is APP or is retrogradely transported shortly after reaching the terminus. A single 150-kDa APLP2 species containing the Kunitz protease inhibitor domain is the major amyloid precursor superfamily protein transported to the presynapse. Presynaptic APP and APLP2 are sialylated and N- and O-glycosylated, and some also carry chondroitin sulfate glycosaminoglycan and/or dermatan sulfate glycosaminoglycan. The rapid kinetics for turnover of APP and APLP2 predict a sensitive balance of synthesis, transport, and elimination rates that may be critical to normal neuronal functions and metabolic fates of these proteins.

Alzheimer Disease↗

PubMed1998

Cellular prion protein localization in rodent and primate brain.

The presence of an abnormal, protease-resistant form of the prion protein (PrP) is the hallmark of various forms of transmissible spongiform encephalopathies (TSE) which can affect a number of mammalian species, including humans. The normal, cellular form of this protein, PrPc, while abundant in brain is also present in many tissues and a number of species. In order to address the unresolved question of the precise localization of normal cerebral PrPc, we used a free-floating immunohistochemistry procedure to localize the protein at both the light and the electron microscopic levels in the brain of three TSE-sensitive species: hamster, macaque and humans. This method shows that PrPc is abundant in synaptic terminal fields in olfactory bulb, limbic-associated structures and in the striato-nigral complex, whereas many other regions of the hamster brain are essentially devoid of immunoreactivity. With the striking exception of the olfactory nerve, in which axons are continually growing throughout life, PrPc is not abundant in fibre pathways. PrPc distribution in the primate hippocampus and cortex is very similar to the distribution observed in hamster. PrPc was present at synaptic profiles as shown by immunoelectron microscopy, but was not detectable in neuronal perikaryon either by light or electron microscopy. Our results show that PrPc is abundant in a number of brain structures known for ongoing plasticity, and are consistent with the hypothesis that the protein also plays a role in synaptic function.

Adult↗

PubMed1997

Developmental shift of synaptic vesicle protein 2 from axons to terminals in the primary visual projection of the hamster.

Synaptic vesicle protein 2 is an integral synaptic vesicle membrane glycoprotein which is present in all synapses for which it has been examined. We used an anti-synaptic vesicle protein 2 monoclonal antibody to examine synaptic vesicle protein 2 localization in the developing hamster retinofugal pathway. From postnatal day 0 to day 1, a period of elongation of retinal ganglion cell axons to their central targets, fiber fascicles in the optic tract over the lateral geniculate nucleus were intensely synaptic vesicle protein 2-immunoreactive. Adjacent to the optic tract, single fibers could be seen. We also observed a marked immunostaining in growth cones and fiber fascicles in retinal explants in culture. By postnatal day 2, the staining of single fibers had ended, and by postnatal day 5, during the formation of terminal arbors, numerous fine puncta of synaptic vesicle protein 2 immunoreactivity were distributed within the neuropil of the lateral geniculate nucleus. In the adult, the optic tract was devoid of synaptic vesicle protein 2 staining, while the neuropil contained distinct immunoreactive profiles, particularly in the outer shell of the lateral geniculate. These synaptic vesicle protein 2-positive profiles closely resembled the grape-like clusters and large swellings of two known retinal axon terminal types. Eye removal resulted in the rapid disappearance of these synaptic vesicle protein 2-labelled terminal profiles contralateral to the enucleation. A similar pattern of synaptic vesicle protein 2 immunoreactivity was observed in the superior colliculus. From postnatal day 0 to day 2, retinal fiber fascicles in the stratum griseum superficiale/stratum opticum were darkly stained for synaptic vesicle protein 2. By postnatal day 5, the immunoreactivity shifted to the neuropil and from postnatal day 6 onwards, the synaptic vesicle protein 2 immunoreactivity was more intense in the stratum griseum superficiale than in the optic fibre layer. This study demonstrates dense synaptic vesicle protein 2-labelling of elongating axons both in vivo and in vitro. However, coincident with the transition from retinal ganglion cell axon elongation to terminal arborization, synaptic vesicle protein 2 is progressively restricted to synaptic terminals and becomes undetectable in axons. This study is the first to document an axonal localization of synaptic vesicle protein 2 during development and raises the question as to its role during axonal elongation.

Animals↗

PubMed1996

Differential synaptic vesicle protein expression in the barrel field of developing cortex.

The distribution of four proteins associated with synaptic vesicles, SV2, synaptophysin, synapsin I, and rab3a, was investigated during postnatal development of the posteromedial barrel subfield (PMBSF) in the rat somatosensory cortex. A distinct progression in the appearance of the different synaptic vesicle proteins within the PMBSF was observed. SV2, synapsin I, and synaptophysin revealed the organization of the barrel field in the neonate. This early demarcation of the cortical representation of the vibrissal array coincides with the earliest known age for the emergence of the cytoarchitectonic organization of this region. In contrast, rab3a did not delimit the barrels until the end of the 1st postnatal week, coincident with the known onset of adult-like physiological activity and the loss of plasticity in afferents to this region. In addition, the appearance of the different synaptic vesicle proteins occurred earlier within the PMBSF than in the adjacent extra-barrel regions of the cortex. These results show that the molecular differentiation of synaptic fields across the cortex is not a homogeneous and synchronous process in terms of synaptic vesicle protein expression. Because these proteins act together in mature synapses to ensure the regulated release of neurotransmitters, our results suggest that this temporo-spatial asynchrony may underlie different potentials for synaptic activity and thus contribute to the development of cortical maps.

Animals↗

PubMed1995

Differential effect of functional olfactory deprivation on synaptic vesicle proteins in rat olfactory bulb.

The effects of functional odour deprivation on three different proteins associated with the membrane of the synaptic vesicle were examined in the rat olfactory bulb. Six weeks after neonatal unilateral nostril closure, Rab3a, a ras-like GTPase, was down-regulated in the odour-deprived bulb in the same manner as tyrosine hydroxylase. In contrast, synaptophysin, a protein of the channel family, and SV2, a putative transporter protein, were not altered. These results suggest that afferent activity is a factor controlling the level of some, but not all, proteins associated with presynaptic vesicles.

Animals↗

PubMed1994

The amyloid precursor protein is developmentally regulated and correlated with synaptogenesis.

Metabolism of the amyloid precursor protein (APP) may contribute to the molecular changes observed in Alzheimer's disease, but the function of the protein in the non-pathologic nervous system remains unknown. In vitro studies have suggested that APP can participate in cellular adhesion and may thus contribute to neuronal differentiation in cultured cells. Here we show, in the primary visual pathway of the hamster, that APPs are developmentally regulated proteins rapidly transported to the growing tips of nerve fibers. Transmembrane forms of higher molecular weight (120 and 140 kDa) are preferentially associated with the rapid elongation of axons. Interestingly, another full-length form of 110 kDa and a soluble form of 100 kDa which lacks the C-terminal domain increase at the time of end-arbor formation and synaptogenesis and then decline when mature connections are established, suggesting that target recognition and synaptic contact may result in a signal for APP cleavage in the CNS in vivo.

Age Factors↗

PubMed1994

Developmental changes in the localization of the synaptic vesicle protein rab3A in rat brain.

Rab3A is a protein associated with the membrane of synaptic vesicles and is involved in the control of the targeting or docking of these vesicles at the presynaptic membrane for the release of neurotransmitters. Here, we have examined the expression and localization of this protein during the development of the rat brain. Relative to total protein, the concentration of rab3A greatly increased during brain development. Both the intracellular localization of the protein and its cerebral distribution showed an age-dependent shift. In contrast to other synaptic vesicle proteins, rab3A was heavily concentrated in cell bodies when immature neurons were migrating and during early differentiation. Later, the protein disappeared from perikarya and had a diffuse distribution in the neuropil, indicating a redistribution to nerve terminals, its exclusive localization in the adult. In the developing somatosensory cortex, rab3A delimited the modular organization of the barrels well after the afferents have arrived but just around the time that mature synaptic activity has been observed. In the hippocampus, rab3A defined a novel "blob-like" organization of the mossy fibre terminals and its appearance in terminal fields closely preceded the known onset of long-term potentiation. The appearance of rab3A in specific terminal fields during the period of increased physiological activity suggests that this small GTP-binding protein may be an important late element in the establishment of the mature characteristics of the presynaptic terminal.

Aging↗

PubMed1994

Identification of a peripherin dimer: changes during axonal development and regeneration of the rat sciatic nerve.

Western blotting of rat dorsal root ganglion (DRG) and sciatic nerve under nonreducing conditions revealed that a peripherin-specific antibody recognized a protein species of 116/130 kDa, pI 5.6, in addition to peripherin (56 kDa, pI 5.6). We showed that this 116/130 kDa protein is a disulfide dimer of peripherin, because it gave rise to a single protein band comigrating with peripherin under reducing conditions and yielded the same proteolytic pattern as peripherin upon N-chlorosuccinimide digestion. In addition, the immunological characteristics of the resulting peptides were identical to those of peripherin. We investigated the changes in peripherin monomer and dimer protein levels during axonal development and regeneration. During postnatal development, quantitative analysis of western blots of DRG proteins showed a significant increase in peripherin monomer (+52%) and dimer (+33%) levels from the day of birth [postnatal day 0 (P0)] to P7. The monomer levels remained high until P14 and then decreased so that at P21 and later ages, the monomer levels were similar to those observed at birth. In contrast, the dimer levels decreased continuously after P7, and in the adult, its level represented only 30% of the level at birth. Changes in [35S]methionine incorporation into adult DRG proteins were studied during regeneration of axotomized sciatic axons. Quantitative analysis of proteins showed a strong increase in labeling of both peripherin monomer (+56%) and dimer (+88%) 7 days after the crush. These levels, which remained high until 28 days after the axotomy, had returned to normal 70 days post axotomy. Our results show that peripherin monomer and dimer greatly increase during DRG fiber development and regeneration, suggesting that the two forms are involved in the growth of axons.

Aging↗

PubMed1994

In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

Amyloid beta-Protein Precursor↗

PubMed1994

Amyloid precursor protein in cortical neurons: coexistence of two pools differentially distributed in axons and dendrites and association with cytoskeleton.

Embryonic cortical neurons in culture contain transmembrane amyloid precursor protein (APP) capable of associating with the detergent-insoluble cytoskeleton through interactions requiring the presence of its C-terminal. These transmembrane APPs are not detectable at the surface of living cells. When neurons are fixed with paraformaldehyde alone, APP is mainly visualized close to the membrane of the axon and cell body of 40% of neurons, with virtually no dendritic staining. Membrane permeabilization with detergent or methanol extends APP immunostaining to 100% of the cells and to all compartments, including the dendrites. Taken together, these results suggest that APP in embryonic neurons is present in two compartments, one more readily detectable in some axons and cell bodies and the other distributed throughout all neurons. The axonal and somatic pool of APP detectable after paraformaldehyde fixation alone is highly and rapidly augmented after exposure to calcium ionophores. We propose that calcium entry increases the amount of axonal APP close to the cell surface, but that the stabilization of the protein at the cell surface and its subsequent secretion require further physiological stimuli.

Amino Acid Sequence↗

PubMed1992

Localization of the ras-like rab3A protein in the adult rat brain.

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e., dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, regions of cerebral cortex and the cerebellum exhibited striking laminar distributions of rab3A immunoreactivity. In the hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentate granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral cortical hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.

Animals↗

PubMed1992

Changes in rapidly transported proteins associated with development of abnormal projections in the diencephalon.

The development of the hamster visual system is accompanied by striking changes in the pattern of proteins that are synthesized in retinal ganglion cells and rapidly transported to their nerve terminals. To determine whether any of these protein changes are regulated by interactions between the developing nerve endings and the cells with which they form synapses, we induced retinofugal axons to form abnormal projections in the lateral posterior (LP) nucleus of the thalamus and dense patches of hyperinnervation in the lateral geniculate nucleus (LGN) by removing their principal target, the superior colliculus (SC), the day after birth. Under these experimental conditions, two rapidly transported proteins, including the neural cell adhesion molecule, NCAM, showed significant changes in their time course of expression. NCAM, identified here using a monospecific antibody, is normally synthesized and transported at high levels at early stages of development and then declines during the second and third postnatal weeks. However, this decline was delayed when optic fibers were re-routed. A second rapidly transported protein, M(r) = 67 kDa, pI = 4.7, normally shows a rise in its synthesis and transport during terminal arbor formation and a subsequent decline, but it also remained elevated for a prolonged period when the SC was absent. These findings cannot be accounted for by a simple delay in the retinal ganglion cells' program of axonal growth, since other rapidly transported proteins, including the growth-associated protein GAP-43, showed a normal developmental time-course when the SC was removed. Target interactions therefore appear to influence the retinal ganglion cells' expression of different proteins in a specific fashion.

Aging↗