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Toward understanding of the molecular basis of loss of neuronal plasticity in ageing.

Loss of neuronal plasticity in ageing

Mature neurons in both the central and peripheral nervous systems of mammals are generally considered postmitotic, and, therefore, cannot be replaced if they die during adult life. It is also believed that age-related cell loss occurs in some but not all regions of the brain, and that neuronal loss could be the cause of some of the functional deterioration that occurs during human ageing |1~. A recent notion, however, is that there is little or no neuronal loss but rather neuronal shrinkage or atrophy in many areas of the brain. The most clear evidence is that in the human neocortex, numbers of large neurons decrease, while those of small neurons increase during ageing, which suggests neuronal shrinkage |2, 3~. It is, therefore, of importance to understand the molecular mechanisms of how neurons lose their cell bodies, axons and dendrites and, ultimately, lose synapses during ageing. A main issue here is neuronal plasticity in ageing, which is a focus of recent research |4~.

The deterioration in brain function that occurs during ageing may be due, at least in part, to a loss of neuronal plasticity. There is evidence that neuronal plasticity does decline with advancing age. For example, in aged rodents, impairments in learning and memory have been associated with an age-dependent decline of cholinergic function in the basal forebrain |5~, and these cholinergic neurons undergo age-dependent atrophy as well |6, 7~. This atrophy in aged rats can be ameliorated, at least in part, by nerve growth factor (NGF) |7, 8~. This observation suggests that neurotrophic factors may overcome some age-related deficits in neuronal function. It also suggests that neurons will suffer a progressive damage if they lose ability to respond to neurotrophic factors which support the survival of various central neurons (for review, see |9, 10~).

There is further evidence which suggests that neuronal plasticity decreases during ageing. For example, Adler and Black pointed out that there may be an age-related change in neurotransmitter plasticity |11~. They examined the synthesis of substance P in explants of sympathetic neurons of superior cervical ganglia (SCG) which is induced by short-term cultures. A tenfold increase of substance P was observed in the explant cultures of ganglia from both neonatal and 6-month-old rats, while no significant increase was observed in 2-year-old rats, suggesting that remarkable plasticity persists in mature neurons while it is deficient in aged sympathetic neurons.

SCG neurons are lineally related to adrenal medullary chromaffin cells, i.e. endocrine cells which synthesize and secrete catecholamines |12~. Indeed, chromaffin cells can undergo a morphological transdifferentiation into sympathetic neuron-like cells, in response to NGF both in vivo and in vitro, a feature known as chromaffin cell plasticity |13~. The transdifferentiation induced by NGF is partially inhibited in the presence of glucocorticoid (GC).

It is of interest to note that the opposing effects of NGF and corticosterone, a peptide growth factor and a glucocorticoid steroid hormone (GC), respectively, are also observed commonly in other systems in relation to cell death. In the brain, hippocampal neurons exhibit trophic effects of NGF and other related neurotrophic factors, while chronic exposure to high GC induces death of certain hippocampal neurons |14~. Although the mechanisms of GC effects may be different in detail |15~, GC also triggers apoptosis, the rapid cell death of lymphocytes in the thymus |16~. Thus, the chromaffin cell plasticity is analogous to neuronal degeneration of hippocampal neurons and apoptosis of lymphocytes. The chromaffin cell system has a distinct advantage over the use of cells from the brain because it is relatively easy to prepare pure populations of cells in sufficient quantity for molecular analysis. Therefore, the chromaffin cell is a good model system to study the molecular basis for the NGF-dependent neuronal plasticity.

There is evidence to suggest that the plasticity of chromaffin cells declines during development, which may overlap or be the same as early stages of ageing |17, 18~. In comparing the NGF-mediated induction of tyrosine hydroxylase (TH) activity and of neurite outgrowth in cultures of adrenal chromaffin cells from fetal, neonatal, and adult bovine animals, Naujoks et al. reported that the neurite outgrowth was observed only in chromaffin cells obtained from fetuses up to a gestational age of 3 months, not in cultures from young adults |17~. Likewise, cultures from fetal and neonatal animals showed a TH increase, not seen in cultures from adult animals. The affinity and density of the low-affinity NGF-receptor (NGF-R), however, did not change from neonate to adult. Unsicker et al. examined neurite extension of chromaffin cells in response to NGF and CNTF (ciliary neurotrophic factor) using rats. Cells from 30-day-old rats extended neurites in response to these factors, while those cells from 100-day-old rats did not |18~. Both studies have thus shown that chromaffin cells of adult animals lose at least some responsiveness to NGF. This raises the questions of how much of the plasticity remains in later stages of ageing, and what mechanisms are involved in this loss of chromaffin cell plasticity.

Neuronal growth associated proteins as molecular markers of neuronal plasticity

Changes in neuronal structure involve inductive gene regulation of neuronal growth-associated proteins (nGAPs), which include neural-specific molecules such as neurofilaments, peripherin, MAP-2, tau, GAP-43 and SCG10. The overall expression profiles of these nGAPs are in some ways similar in that they are expressed at high levels during neurogenesis in embryonic and postnatal periods and low but significant levels of expression still persist into adulthood. Thus, the expression of nGAPs is closely correlated with neurite elongation during neurogenesis, and it also seems to contribute to synaptic modification in the adult brain.

Most nGAPs are phosphoproteins, whose levels of phosphorylation change in response to extracellular signals such as NGF or in neurodegenerative conditions such as in Alzheimer's disease. In addition, nGAPs are known to interact with cellular membranes and cytoskeletal components. For example, GAP-43 and SCG10 are membrane-associated molecules |19, 20~, while MAP-2 and tau are associated with microtubules |21~. Neurofilaments and peripherin interact with vimentin |22, 23~.

The amino acid sequences of nGAPs are distinctly different from each other suggesting different roles in cellular function as well as different anatomical distribution within the cell. Indeed, some nGAPs present only in dendrites, and others in axons. mRNA of MAP-2A and MAP-2B is detected predominantly in dendrites but not in axons |24~, while tau and GAP-43 are localized in axons of mature neurons |25~. Thus, some nGAPs show unique spatial distributions, even though the timing of the expression is quite similar in general. Several nGAPs share structural and functional similarities. For example, GAP-43 and neurogranin share similar amino acid sequences in a region which corresponds to the calmodulin-binding domain |26~.

We have been studying one of these nGAPs, SCG10, which was originally isolated from a cDNA library of superior cervical ganglia as clone no. 10 |27~. The expression of SCG10 is widely observed in developing central and peripheral neurons, and is particularly characteristic in the migrating neural crest populations which later form the peripheral nervous system |20~. SCG10 is expressed also in mature chromaffin cells and PC12 cells at low level and is inducible by NGF |20, 27~. The induction occurs at least 20 h after the addition of NGF, and requires new protein synthesis |28~, which probably involves a set of transcriptional factors such as NGF-IA/Zif268 and c-fos, the so-called immediate early genes |29~. The expression of SCG10 is correlated with neuronal differentiation of chromaffin cells and PC12 cells, in which it is induced by NGF and is partly inhibited by the presence of GC |28~. Therefore SCG10 serves as a molecular marker of chromaffin cell plasticity, in which non-process bearing adrenal chromaffin cells can be reversibly converted into adrenergic neurons |13~. SCG10 may also be regarded as a marker of neuronal plasticity since it is induced following a partial lesion in peripheral ganglia (see the discussion in |30~).

SCG10 is a neural-specific protein consisting of 179 amino acid residues |20~, which shares homology with another neural-enriched (but not neural-specific) protein called p19 or stathmin |31, 32~. Overall amino acid identity is 69%. A distinct difference is that p19/stathmin lacks the hydrophobic N-terminal domain which presents in SCG10 and seems to contribute to its interaction with membranes. While the functions of SCG10 and p19/stathmin are not clear at the moment, Sobel et al. proposed that stathmin may have a role in signal transduction mechanisms |33~, since levels of phosphorylation of stathmin by a cAMP-dependent protein kinase (A-kinase) change rapidly in response to extracellular stimuli such as NGF |34~. Three serine residues which are the potential phosphorylation sites are all conserved in p19/stathmin and TABULAR DATA OMITTED SCG10, thus SCG10 is also probably a phosphoprotein.

Another nGAP, GAP-43, is also a phosphoprotein, but is a substrate for protein kinase C (C-kinase) |19~. The presence of two unique membrane-associated phosphoproteins in growth cones is interesting in light of studies linking intracellular calcium ion and other second messengers to growth cone behaviour involved in synaptic remodelling in adult neurons. Phosphorylation of specific proteins is known to be involved in synaptogenesis and remodelling in the hippocampus, the structure which seems involved in memory function |35~.

Memory loss is a frequently occurring functional deficit of the ageing human brain. The underlying mechanisms of age-related memory loss may be one of the most complicated questions in biology, and is also an unshirkable challenge for ageing research. To approach this problem, it seems to be of importance to know the expression and regulation of molecules of neuronal growth control, i.e. nGAPs, in brains in normal and diseased conditions. The regulatory mechanisms of nGAP expression could be studied directly in the brain, or by using chromaffin cells and PC12 cells as a model system of neuronal plasticity.

Expression of SCG10 in the brain during development, maturation and ageing

The expression of SCG10 is particularly characteristic in the neuronal subpopulations of migrating neural crest derivatives which later form the peripheral nervous system including the dorsal root ganglia and the superior cervical ganglia. However, the expression of SCG10 is also evident in the ventral spinal cord in developing embryos |20, 27~, suggesting that SCG10 is expressed in the central nervous system as well.

The brain expression of SCG10 mRNA was examined by in situ hybridization to sagittal sections of adult rat brains. The SCG10 expression is maximal in mid to late gestation of the embryo. Fairly intense expression occurs in wide areas of neonatal and immature brains (not shown), followed by a dramatic decrease in the adult. However, the expression still persists into the adult at low and significant levels in many areas of the brain. Positive areas with the highest level of SCG10 in the adult brain include the mitral cell layer of the olfactory bulb, the piriform cortex, the hippocampus, and the Purkinje and granular cells of the cerebellum. Moderate signals were found in the large pyramidal cell layer of the cerebellar cortex, and in several nuclei in the thalamus and brain stem. Details of the in situ hybridization experiments will be published elsewhere (T. Himi et al., in preparation).

The distribution of SCG10 proteins was examined by immunohistochemistry using antibodies against SCG10. The results were similar to that of the in situ hybridization experiments, although the SCG10 protein was distributed in wider areas, probably owing to transportation along dendrites and axons (Y. Sugiura et al., unpublished).

The distribution of SCG10 is of interest, because the brain areas where the higher expression was observed in the adult are known to maintain neuronal plasticity in adults. These results suggest that SCG10 may be involved in the remodelling of synaptic connections in response to various intrinsic and extrinsic signals in adult brains. Indeed SCG10 was shown to be induced in the contralateral cortex following unilateral striatal deafferentiation |36~.

Is the expression of SCG10 maintained in the later stages of life? In order to answer this question, we quantitated SCG10 mRNA levels during ageing in both the brain and the adrenal tissues. RNase protection assays indicated that the SCG10 expression in olfactory bulbs of 24-month-old rats decreased by 50% in comparison with 6-month-old rats, while in the hippocampus, such a reduction of SCG10 mRNA was not observed. However, in preliminary in situ hybridization experiments using the hippocampus tissues of the young and old animals, there seemed a difference in the expression profiles between the young and old animals (not shown). Young hippocampal tissues showed an apparent regional expression of SCG10 mRNA, i.e. higher expression in pyramidal cells of the CA3-CA4 region and lower expression in granular cells of the dentate gyrus, while old hippocampi did not show such a clear regional distribution, which suggests that the regulation of SCG10 expression may be impaired in the old brain. However, the possible changes in the regional distribution of SCG10 need to be confirmed by further experiments.

These results with SCG10 suggest that the expression of other nGAPs may also change during ageing. Both the examination of the expression profiles of nGAPs during ageing and the exploration of regulatory mechanisms of those genes will contribute to the understanding of molecular mechanisms of the loss of neuronal plasticity in the ageing brain.

Molecular studies of neuronal plasticity using cells of the sympathoadrenal lineage

As mentioned above, adrenal medullary chromaffin cells offer a good model system to study the molecular mechanisms of neuronal plasticity. PC12 cells, phaeochromocytoma, are also used for this purpose. Recent studies indicate that PC12 cells express trkA, a high-affinity NGF-receptor |37, 38~. Thus the trkA-expressing PC12 cells and chromaffin cells are in good contrast with central neurons expressing trkB or trkC corresponding to BDNF or NT-3 (see |9, 10~ for reviews). The downstream events following the tyrosine phosphorylation of the trk receptors are believed to be very similar, which also suggests the equivalence of the chromaffin cell plasticity and neuronal plasticity.

As a first step to investigating how much plasticity remains during ageing, we examined the basal expression of SCG10 in adrenal tissues of rats of various ages. Low but significant levels of SCG10 expression were observed throughout the lifespan up to 24-month-old in both tissues. Although there seems a slight increase in the basal expression of the SCG10 gene after maturation, it is not clear whether it is due to the changes of extrinsic factors (peptidergic growth factors or GC) in the adrenal gland during maturation and ageing.

To approach the molecular mechanisms of how NGF induces nGAPs, we examined whether the induction of SCG10 by NGF involves transcriptionally inducible cis-regulatory elements in the SCG10 gene |39-41~. Transfection experiments using various SCG10-CAT fusion constructs in PC12 cells revealed that the NGF-inducible elements resided, at least in part, in the distal but not the proximal regulatory region of the SCG10 gene. The significant induction of CAT activity was observed only in assays using stably transformed PC12 cells, while transient transfection assays showed ectopic induction with all kinds of promoters examined (also see discussion in |40~).

These results indicate that, first, the expression of SCG10 is maintained at a basal level even at old ages in adrenal tissues, suggesting that the SCG10 gene in chromaffin cells of old animals is accessible to the exogenous stimuli which can induce this gene. Second, at least some of NGF-inducible transcriptional cis-regulatory elements are present in the 4 kb upstream sequence of the SCG10 gene. Characterization of the NGF-responsive element of the SCG10 gene and comparing it with that of other NGF-inducible nGAPs will give the basis for understanding the mechanism of the induction of neuronal phenotype by NGF. It is also of interest to see the relationship or its possible interaction with the other cis-regulatory elements so as to determine neuronal cell-specific expression of the SCG10 gene. Since we generated a series of deletion and addition constructs of SCG10-CAT fusion plasmids and precisely mapped a cis-regulatory silencer element which primarily determined the neural specific expression (submitted for publication), those constructs will be useful materials to explore the NGF-regulatory elements in the SCG10 gene.

As discussed above, the NGF-responsiveness of chromaffin cells decreases during ageing. The reason for the loss of NGF-responsiveness has not yet been understood. It could be caused by the loss of functional NGF-receptors, the defects in secondary signalling pathways, or the loss of specific transcriptional factors. There is evidence, in experiments using rat chromaffin cells, that the density of NGF-receptors does not change between newborn and 10-day-old rats (10-22 000 receptors per cell) |42~. It is of importance to examine whether this is also true for later stages of life. There might be changes in the efficacy of secondary messenger pathways which follow the activation by the binding of NGF to trk on the chromaffin cell surface. Alternatively, there might be a loss of DNA-binding proteins which regulate the transcription of neural-specific genes which are induced by NGF. In this regard, it is of interest to search cis-regulatory element(s) in the SCG10 gene, identify DNA-binding factor(s) for those elements, and examine whether the levels of the factors may decrease in chromaffin cells during ageing.

Concluding remarks

Understanding the molecular mechanisms of the loss of neuronal plasticity during ageing is an important task for gerontological research. By choosing an appropriate model of neuronal plasticity such as chromaffin cells, and focusing on marker molecules of neuronal growth, nGAPs, I hope that some clues will be found which may contribute to the prevention of neurodegenerative conditions which frequently affect elderly people.

In ancient oriental culture, the pictographic character of 'senescence' (ROW in Japanese, LAO in Chinese) is an image of an old man with hair dishevelled, bending to walk with a stick. It is my long-cherished wish to transform this image into a character SEI (in Japanese, SHENG in Chinese) that means 'life' or 'to live', or into JU (in Japanese, SHOU in Chinese) that means 'happiness with longevity', by manipulating those scattered filaments in the old man's head. Indeed, to understand the control mechanisms of neuronal growth seems an important subject as well as a challenge for ageing research in following decades.

Acknowledgements

I thank the following individuals in my laboratory, who have contributed to the work described in this review: Haimei Wang, Drs Toshiyuki Himi, Yoshie Sugiura, and Takashi Okazaki. I also wish to thank Dr David J. Anderson (Caltech) in whose laboratory the basis of this research was initiated. I am grateful to Drs. Caleb E. Finch and Thomas N. McNeill for comments on the manuscript and Minghua Cao for assistance in preparation of the manuscript. This work was supported by grants from the Sandoz Foundation for Gerontological Research, Alzheimer's Association, Max Factor Foundation and National Institute on Aging (AG 07909). This paper is dedicated to the memory of Dr David H. Mitchell.

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Author:Mori, Nozomu
Publication:Age and Ageing
Date:Jan 1, 1993
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