Modeling cholinergic abnormalities in Down syndrome and Alzheimer's disease.
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Alzheimer Disease↗
Biomedical subjects
W C Mobley
Publications and source records attributed to W C Mobley.
Regulation of axonal caliber, neurofilament content, and nuclear localization in mature sensory neurons by nerve growth factor.
The neuronal perikaryal response to axonal injury (axon reaction) includes reduction in axonal caliber beginning in the proximal portion of the nerve (somatofugal axonal atrophy), development of nuclear eccentricity, and chromatolysis. The means by which these events are triggered is unknown, but it has been argued that loss of a neurotrophic signal from the target of injured neurons plays a role. To date, the identity of this substance(s) remains unknown. In the present study, we have asked whether NGF normally functions to control axonal caliber of sensory neurons in the L4 and L5 dorsal root ganglia (DRG) of the adult rat. Two approaches were used: (1) NGF was continuously delivered to the proximal stump of a transected sciatic nerve to determine whether NGF administration would prevent the production of somatofugal axonal atrophy; and (2) NGF antisera were administered to normal animals to determine whether NGF deprivation would produce somatofugal axonal atrophy. In the first experiment, 9-week-old rats underwent a unilateral sciatic nerve transection at midthigh, and the proximal stump was connected to an osmotic pump containing either NGF or cytochrome C (as control). At 11 weeks of age, dorsal root fibers in lumbar DRG from the control group appeared smaller in caliber and less circular in shape than fibers from age-matched normal animals. Although smaller than those in normal animals, fibers from the NGF-treated nerves were larger than in axotomized controls. Mean axonal area and shape factor (an index in circularity) were measured and found to be decreased significantly (22% and 15%, respectively) from the control group. Fibers from the NGF-treated nerves were significantly (p less than 0.05) larger in axonal caliber and more circular in shape; mean values were only reduced by 11% and 10%, respectively. Quantitation of neurofilament (NF) numbers revealed that the larger calibers in the NGF-treated nerves result from a greater NF content. NGF treatment did not prevent the atrophy of motor fibers in the proximal ventral root. In the second experiment, 2 antisera to mouse NGF were given daily into the footpad for 11 or 12 d; control animals were given normal goat serum. Quantitation of axonal calibers in the L5 DRG demonstrated that mean axonal area and shape factor were significantly (p less than 0.05) reduced by 14% and 17% respectively. The axoplasm of atrophic fibers demonstrated a paucity of NFs.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The protease inhibitory properties of the Alzheimer's beta-amyloid precursor protein.
We have expressed the 57-amino acid Kunitz domain of the Alzheimer's beta-amyloid precursor protein (APP751) as a bacterial fusion protein. The protease inhibitory properties of the purified fusion protein, BX9, were virtually identical in all respects tested to those of purified secreted APP751. Both proteins strongly inhibited pancreatic trypsin (Kis = 0.2 and 0.3 nM) and less well epidermal growth factor-binding protein (Kis = 1 and 3.5 nM), alpha-chymotrypsin (Kis = 3 and 6 nM), and the gamma-subunit of nerve growth factor (Kis = 8 and 9 M). Neither protein appreciably inhibited plasma and pancreatic kallikreins, thrombin, lung tryptase, neutrophil elastase, or cathepsin G. The remarkable similarity of the protease inhibitory profile of BX9 to that of secreted APP751 suggests that proper intramolecular disulfide bond formation has occurred in the bacterial fusion protein and leads to the conclusion that the amyloid precursor protein Kunitz domain is a relatively specific inhibitor of only a few trypsin-like arginine esterases.
Amino Acid Sequence↗
Trophic actions of recombinant human nerve growth factor on cultured rat embryonic CNS cells.
NGF is a neurotrophic factor for basal forebrain cholinergic neurons and may serve to counteract the cholinergic deficits that are observed in Alzheimer's disease. Prior to the introduction of clinical trials, it is essential that recombinant human NGF (rhNGF) be produced and that its actions on target cells in the CNS be demonstrated. We prepared rhNGF and examined its actions on fetal rat brain neurons in culture including, in particular, the cholinergic neurons of the basal forebrain. rhNGF was more potent in increasing choline acetyltransferase (ChAT) activity in septal cultures than NGF purified from mouse salivary glands (mNGF). ED50s of the beta-NGF dimers were 4.9 pM for rhNGF and 12.4 pM for mNGF. The maximal ChAT activity response was achieved at approximately 35 pM with both NGFs and their efficacies were not significantly different. The two NGFs were not additive in effect. Identical to the results with mNGF, rhNGF strongly enhanced the intensity of ChAT immunostaining in septal cultures. Neither rhNGF nor mNGF affected the appearance of the cultures under phase-contrast illumination. Survival of cells at very low plating density on polyornithine/laminin-coated culture dishes was not affected by rhNGF or mNGF. Protein content and the uptake of GABA were also unaffected. At concentrations of up to 10 micrograms/ml, rhNGF did not significantly increase uptake of dopamine into cultures of ventral mesencephalon. We conclude that rhNGF produces potent and selective actions on cholinergic neurons of the basal forebrain as previously shown for mNGF.
Animals↗
Nerve growth factor induces gene expression of the prion protein and beta-amyloid protein precursor in the developing hamster central nervous system.
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Amyloid beta-Peptides↗
The in vitro biological effect of nerve growth factor is inhibited by synthetic peptides.
Nerve growth factor (NGF)1 is a neurotrophic polypeptide that acts via specific receptors to promote the survival and growth of neurons. To delineate the NGF domain(s) responsible for eliciting biological activity, we synthesized small peptides corresponding to three regions in NGF that are hydrophilic and highly conserved. Several peptides from mouse NGF region 26-40 inhibited the neurite-promoting effect of NGF on sensory neurons in vitro. Inhibition was sequence-specific and could be overcome by increasing the concentration of NGF. Moreover, peptide actions were specific for NGF-mediated events in that they failed to block the neurotrophic activity of ciliary neuronotrophic factor (CNTF) or phorbol 12-myristate 13-acetate (PMA). In spite of the inhibition of NGF activity, peptides did not affect the binding of radiolabeled NGF. These studies define one region of NGF that may be required for neurotrophic activity.
Amino Acid Sequence↗
In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain.
Oligonucleotide DNA probes were used to determine the distribution of mRNAs encoding the alpha- and beta-subunits of Ca2+/calmodulin-dependent protein kinase type II (CaM-KII) in developing rat brain. The regional and temporal distribution of these mRNAs closely paralleled the distribution and developmental appearance previously reported for their respective protein subunits. alpha-Subunit mRNA was barely detectable in sagittal sections at 4 d postnatal but increased as much as 10-fold in frontal cortex by day 16. beta-Subunit mRNA, on the other hand, was readily detected at 4 d postnatal and changed only slightly during development. Telencephalic structures exhibited the highest levels of CaM-KII mRNA and the brain stem displayed the least. alpha-Subunit mRNA was not observed in cerebellar granule cells and was barely detectable in Purkinje cells, while the beta-mRNA was easily detected in both neuronal types. mRNAs for both alpha- and beta-subunits were present in many neuronal cell bodies; however, only the alpha-subunit mRNA was localized to molecular layers of the hippocampus and lamina I of the frontal cortex. These layers of neuropil are relatively cell sparse and contain extensive dendritic arborizations and synaptic contacts. Since polyribosomes have been observed near hippocampal dendritic spines, the localization of alpha-subunit mRNA to dendrites of pyramidal and dentate granule cells suggests that this subunit is synthesized in situ at postsynaptic sites. The co-localization of translational machinery and high concentrations of CaM-KII in postsynaptic elements suggests an important relationship between alpha-subunit synthesis and the maintenance and plasticity of postsynaptic structures.
Animals↗
Mouse nerve growth factor prevents degeneration of axotomized basal forebrain cholinergic neurons in the monkey.
NGF, a trophic polypeptide, is necessary for the normal development and survival of certain populations of neurons in the CNS and PNS. In the CNS, cholinergic neurons of the basal forebrain magnocellular complex (BFMC) are prominent targets of NGF. During rat development, NGF increases the activity of ChAT in these neurons. In adult rats with experimental injury of axons in the fimbria-fornix, NGF prevents degenerative changes in axotomized cholinergic BFMC neurons in the medial septal nucleus (MSN). Because the amino acid sequences of NGF and its receptor (NGF-R) are highly conserved across species, we hypothesized that mouse NGF would also prevent degeneration of cholinergic BFMC neurons in nonhuman primates. Therefore, the present study was designed to test whether fimbria-fornix lesions result in retrograde degenerative changes in basal forebrain cholinergic neurons in macaques, whether these changes are prevented by mouse NGF, and whether the protective effect of NGF is selective for cholinergic neurons of the basal forebrain. Following unilateral complete transection of the fornix, animals were allowed to survive for 2 weeks, during which time half of the subjects received intraventricular NGF in vehicle and the other half received vehicle alone. In animals receiving vehicle alone, there was a 55% reduction in the number of ChAT-immunoreactive cell bodies within the MSN ipsilateral to the lesion; loss of immunoreactive somata was more severe in caudal planes of the MSN. Remaining immunoreactive neurons appeared smaller than those in control, unoperated animals. In Nissl stains, there was no apparent loss of basophilic profiles in the MSN, but cells showed reduced size and intensity of basophilia. Treatment with NGF almost completely prevented reductions in the number and size of cholinergic neurons and had a significant general effect in preventing atrophy in basophilic magnocellular neurons of the MSN, though some basophilic neurons in the MSN did not appear to respond to NGF. Adjacent 7-microns-thick sections stained with ChAT and NGF-R immunocytochemistry revealed that these markers are strictly colocalized in individual neurons in the MSN in controls and in both groups of experimental animals. Thus, mouse NGF profoundly influences the process of axotomy-induced retrograde degeneration in cholinergic BFMC neurons in primates. The in vivo effectiveness of mouse NGF on primate BFMC neurons suggests that mouse or human recombinant NGF may be useful in ameliorating the ACh-dependent, age-associated memory impairments that occur in nonhuman primates.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholinesterase↗
Intraventricular NGF infusion in the mature rat brain enhances sympathetic innervation of cerebrovascular targets but fails to elicit sympathetic ingrowth.
The ability of peripheral axons to regenerate long distances in the peripheral nervous system (PNS) is well documented; however, examples of axonal elongation within the adult mammalian central nervous system (CNS) are rare. One example of axonal growth in the mature brain is the sprouting of sympathetic axons into the hippocampal formation following disruption of the septohippocampal pathway. A current hypothesis is that elevated hippocampal NGF levels, secondary to loss of retrograde transport by septal neurons, elicits sympathetic ingrowth, In this study, we sought to determine whether elevation of hippocampal NGF activity without septal denervation is sufficient to elicit sympathetic sprouting. Forty-one female rats were infused for two weeks with NGF or cytochrome C in the right lateral ventricle through cannulae connected to an osmotic minipump. In some animals the brains were sectioned and stained for acetylcholinesterase (AChE) activity and norepinephrine histofluorescence; in others, CNS tissue was assayed for nerve growth factor (NGF) content with a two-site ELISA. A Farrand microspectrophotometer was used to measure the intensity of catecholamine fluorescence around the internal carotid artery. The average fluorescence intensity of the sympathetic innervation of the internal carotid artery in the NGF-injected animals was over twice that of vehicle-injected rats indicating that the infused NGF was both accessible to the sympathetic axons and biologically active. However, in none of the cases with elevated hippocampal NGF levels were sympathetic axons observed within the hippocampal formation or any other brain region. These results suggest that simple elevation of brain NGF, while perhaps necessary, is insufficient to permit the growth of sympathetic axons into the mature mammalian CNS.
Adrenergic Fibers↗
Induction of the c-fos gene product in rat forebrain following cortical lesions and NGF injections.
Neocortical lesions and NGF injections into neocortex induce the immunostaining of Fos, the c-fos gene product, in neuronal nuclei in ipsilateral cortex, and amygdala. Adjacent structures including hippocampus, septal nuclei, globus pallidus, and thalamus were unaffected. It is hypothesized that trophic molecules or other chemicals are released at the injury site and these induce the c-fos gene in cells throughout the ipsilateral hemisphere. Fos induction might mediate metabolic or plasticity responses to the focal injury.
Animals↗
The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures.
Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.
Animals↗
Developmental regulation of nerve growth factor and its receptor in the rat caudate-putamen.
In prior studies, nerve growth factor (NGF) administration induced a robust, selective increase in the neurochemical differentiation of caudate-putamen cholinergic neurons. In this study, expression of NGF and its receptor was examined to determine whether endogenous NGF might serve as a neurotrophic factor for these neurons. The temporal pattern of NGF gene expression and the levels of NGF mRNA and protein were distinct from those found in other brain regions. NGF and high-affinity NGF binding were present during cholinergic neurochemical differentiation and persisted into adult-hood. An increase in NGF binding during the third postnatal week was correlated with increasing choline acetyltransferase activity. The data are consistent with a role for endogenous NGF in the development and, possibly, the maintenance of caudate-putamen cholinergic neurons.
Animals↗
Acceleration of scrapie in neonatal Syrian hamsters.
Prions cause Creutzfeldt-Jakob disease, Gerstmann-Sträussler syndrome, and kuru of humans as well as scrapie of animals. Prolonged incubation periods, from months to decades, precede clinical disease. In studies on the biochemical characteristics of prions, weanling Syrian hamsters have been used extensively because they have relatively short incubation periods. In studies reported here, inoculation of neonatal hamsters significantly shortened the scrapie incubation period even further. Our results show that the scrapie incubation period in hamsters is a function of age. The interval between inoculation and death from scrapie plotted as a function of age (0 to 30 days) gave a correlation coefficient (r) of 0.86. The duration of clinical disease was also shortened in the hamsters inoculated as neonates compared with weanlings. Intraventricular injection of nerve growth factor prior to inoculation of neonates with scrapie significantly diminished the acceleration observed with scrapie alone in neonates. Histopathologic studies of brain from scrapie-inoculated neonates showed more extensive neuronal loss in the hippocampus and neocortex as well as a more profound gliosis in the caudate compared with animals inoculated as weanlings. Our results demonstrate an age-dependent acceleration of scrapie in neonatal hamsters and may provide a new experimental system for defining factors that modify the pathogenesis of prion diseases.
Animals↗
PrPSc in scrapie-infected hamster brain is spatially and temporally related to histopathology and infectivity titer.
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Amyloidosis↗
Infertility, chemical exposure, and farming in Iowa: absence of an association.
An increased incidence of infertility and abnormal semen analysis has been described in men and experimental animals exposed to agricultural chemicals. These reports led us to review the records of 382 consecutive new male patients who had semen analyses performed at the University of Iowa Hospitals and Clinics. No significant differences in the incidence of abnormalities in either the physical examination or the semen analysis were discovered with respect to occupation or possible exposure to chemical agents.
Adult↗
Nerve growth factor increases mRNA levels for the prion protein and the beta-amyloid protein precursor in developing hamster brain.
Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the beta protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the beta-protein precursor (beta-PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and beta-PP mRNAs. The increases in PrP and beta-PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and beta-PP mRNA levels. Increased PrP and beta-PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and beta-PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.
Amyloid↗
Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector.
To study posttranslational mechanisms for the control of nerve growth factor (NGF), we used a recombinant vaccinia virus vector to independently express the two major NGF transcripts in a variety of mammalian cell lines. The two major transcripts contain NGF (12.5 kilodaltons [kDa]) at the C-terminus and differ by alternative splicing of an N-terminal exon, so that the large precursor (34 kDa) had 67 amino acids upstream of an internal signal peptide and the smaller precursor (27 kDa) had this signal peptide at its N-terminus. In L929 cells, expression of either NGF transcript with the vaccinia virus vector gave rise to an apparently identical intracellular 35-kDa glycosylated precursor formed by cleavage of the primary gene product after the signal peptide. These cells also secreted biologically active NGF. To determine whether NGF processing is restricted by cell type, we infected a variety of mammalian cell lines with both recombinant viruses; all accumulated the same 35-kDa precursor and secreted NGF. Thus, many types of cells have the machinery to process and secrete NGF. However, NGF accumulated intracellularly (presumably in secretory granules) in cells with a regulated pathway of secretion (e.g., AtT-20 and HIT cells). In these cells, a membrane-permeable cyclic AMP analog, 8-bromo-cyclic AMP, stimulated NGF secretion. This suggests a mechanism for the regulation of NGF levels in which specific secretagogues, e.g., neurotransmitters, control NGF secretion.
Animals↗
Neurological dysfunction after intrathecal injection of dynorphin A (1-13) in the rat. I. Injection procedures modify pharmacological responses.
In rats, the spinal subarachnoid injection of the kappa opioid agonist Dynorphin A (Dyn A)(1-13) and the delta opioid receptor antagonist ICI 174864 produced dose-related flaccid paralysis of hindlimbs and tail that were influenced appreciably by injection procedures. When injected through indwelling intrathecal (i.t.) catheters terminating at L1 to L2, both peptides were significantly more potent producing paralysis 1 day, rather than 10 to 14 days, after i.t. catheterization. Other rats received direct subarachnoid injections of these peptides through 30-gauge needles placed in the L4 to L5 intervertebral space. In naive, uncatheterized and acutely catheterized rats, direct intervertebral injection of these peptides, as well as D-Ala2-Dyn A (1-13) amide (a metabolically stable analog of Dyn A (1-13), produced hindlimb paralysis with potencies comparable to those recorded after injections through acutely implanted catheters. In contrast, chronically catheterized rats showed significantly reduced responsivity to direct intervertebral injections of all three of these peptides. Loss of hindlimb motor function was associated with loss of nociceptive responsiveness. Elevations in tail-flick latencies were only seen with doses of Dyn A (1-13) which produced motor dysfunction, and were not blocked or reversed by high doses of the opioid antagonist naloxone. These results indicate that: 1) indwelling i.t. catheters induce spinal cord alterations which complicate their experimental usefulness, 2) Dyn A (1-13) does not alter responsiveness to thermal nociceptive stimuli through opioid mechanism and 3) Dyn A (1-13) causes parallel disruptions of spinal cord motor and nociceptive function.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗