- Email: [email protected]
Changes in Ca2+-binding proteins in human neurodegenerative disorders
J. Acoust. Soc. Am. 72, 131-141 Pickles, J. O. (1985) Prog. NeurobioL 24, 1-42 Hudspeth, A. J. (1982) J. Neurosci. 2, 1-10 Ohmori, H. (1988)J. Physiol. 399, 115-137 Huang, P. L. and Corey, D. P. (1990) Biophys. J. 57, 530a Jaramillo, F. and Hudspeth, A. J. (1991) Neuron 7, 409-420 Osborne, M. P., Comis, S. D. and Pickles, J. O. (1984) Cell Tissue Res. 237, 43-48 27 Little, K. F. and Neugebauer, D-Ch. (1985) Cell Tissue Res.
21 22 23 24 25 26
242,427-432 28 Furness, D. N. and Hackney, C. M. (1985) Hear. Res. 18, 177-188 29 Osborne, M. P., Comis, S. D. and Pickles, J. O. (1988) Hear. Res. 35, 99-108 30 Neugebauer, D-Ch. and Thurm, U. (1987) Cell Tissue Res. 249, 199-207 31 Pickles, J. O., Rouse, G. W. and von Perger, M. (1991) Scanning Microsc. 5, 111 5-1128 32 Comis, S. D., Pickles, J. O. and Osborne, M. P. (1985) J. Neurocytol. 14, 113-130 33 Rhys-Evans, P. H., Comis, S. D., Osborne, M. P., Pickles, J. O. and Jeffries, D. J. R. (1985) J. Laryngol. Otol. 99, 11-19 34 Rouse, G. W. and Pickles, J. O. (1991) J. NlorphoL 209,
111-120 35 Szabo, T. (1974) in Handbook of Sensory Physiology 3 (Fessard, A., ed.), pp. 13-58, Springer 36 Assad, J. A. and Corey, D. P. J. Neurosci. (in press) 37 Eatock, R. A., Corey, D. P. and Hudspeth, A. J. (1987) J. Neurosci. 7, 2821-2836 38 Howard, J. and Hudspeth, A. J. (1987) Proc. NatlAcad. Sci. USA 84, 3064-3068 39 Shepherd, G. M. G. et al. J. Gen. Physiol. (in press) 40 Stevens, S. S. and Davis, H. (1983) Hearing: Its Psychology and Physiology, Wiley 41 Hillman, D. E. (1969) Brain Res. 13, 407-412 42 Dallos, P. (1973) The Auditory Periphery, Academic Press 43 Hackney, C. M., Furness, D. N. and Benos, D. J. (1991) Scanning Microsc. 5, 741-746 44 Jorgensen, F. and Ohmori, H. (1988) J. Physiol. 403, 577-588 45 Shepherd, G. M. G., Barres, B. A. and Corey, D. P. (1989) Proc. Natl Acad. Sci. USA 86, 4973-4977 46 Gillespie, P. G. and Hudspeth, A. J. (1991)J. Cell Biol. 112, 625-640 47 Shepherd, G. M. G., Corey, D. P. and Block, S. M. (1990) Proc. Natl Acad. Sci. USA 87, 8627-8631
Acknowledgements Work in the authors' laboratories was supported by the MRC of the UK and the Australian ResearchCouncil (J.O.P.)and by the Nab'onallnstitutes of Health, the Office of Naval Research, and the Howard Hughes Medical Institute (D.P.C.).
Changesin Caz +-bindingproteinsin human neurodepenerativedisorders Claus W. Heizmann
The cellular distribution of Cae+-binding proteins has been extensively studied during the past decade. These proteins have proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major roles are assumed to be Ca e+ buffering and transport, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca e+ homeostasis, a protective role for cae+-binding proteins in certain neuron populations has been postulated. As massive neuronal degeneration takes place in several brain diseases of humans, such as Alzheimer's disease, Parkinson's disease and epilepsy, changes in the expression of Ca 2+binding proteins have therefore been studied during the course of these diseases. Although the data from these studies are inconsistent, the detection and quantification of Cae+-binding proteins and the neuron populations in which they occur may nevertheless be useful to estimate, for example, the location and extent of brain damage in the various neurological disorders. I f future studies advance our knowledge about the physiological functions of these proteins, the neuronal systems in which they are expressed may become important therapeutical targets for preventing neuronal death in an array of neurodegenerative diseases. More than 7000 articles on calcium were published in 1991 (a figure found using the Medline database, with 'calcium' as the search word). This emphasizes the tremendous interest and progress in Ca2+-related research. In nerve cells, Ca 2+ ions activate and regulate a number of key processes, including fast axonal transport of substances, synthesis and release of some neurotransmitters, and membrane excitability. It is also thought that in the CA1 hippocampal region of the brain, Caz+ might induce long-term potentiation and memory storage mechanisms. The Ca2+ message is converted into an intracellular response - in many cases by CaZ+-binding proteins TINS, Vol. 15, No. 7, 1992
and Katharina Braun
that are involved in a wide variety of activities, such as cytoskeletal organization, cell motility and differentiation, cell-cycle regulation, and Ca2+ buffeting and transport. It might therefore be possible that altered levels of some Ca2+-binding proteins (e.g. due to deletion or mutation of the corresponding genes) could lead to an impaired Ca2+ homeostasis in nerve cells and to pathological conditions. Using mostly immunohistochemical techniques, several research groups have now started to search for altered expression of the CaZ+-binding proteins parvalbumin, calbindin-D28K and S100 in affected brain regions of patients suffering from acute insults, such as stroke and epileptic seizures, and from chronic neurodegenerative disorders, such as Alzheimer's, Huntington's, Parkinson's and Pick's diseases. In addition, altered Ca2+ levels have been found in platelets of patients with bipolar affective disorders 1, and Ca 2+ antagonists have been suggested for treatment of psychotic depression. The few proteins that have been investigated in these pathological states belong to a large family of more than 200 members. These proteins are characterized by a common structural motif, the EF-hand, which is present in multiple copies and binds Ca2+ selectively and with high affinity. Each of these domains consists of a loop of 12 amino acids that is flanked by two ochelices. This structural principle was first identified as a result of studying the crystal structure of the Ca2+-binding protein parvalbumin isolated from carp, and is designated the EF-hand because of the arrangement of the E and F helices of parvalbumin. A consensus amino acid sequence for this motif has aided the identification of many new members of this protein family2'3. Ca2+-binding proteins with known functions, such as calmodulin, troponin C, myosin light chains, calpain, calcineurin and recoverin, are far outnumbered by those with unidentified roles. Most Ca2+-
© 1992, ElsevierSciencePublishersLtd,(UK)
Claus W. Heizrnannis at the Dept of Pediatrics, Division of Clinical Chemistry, Universityof Zurich, 5teinwiesstr. 75, CH-8032 Zurich, Switzerland,and
Katharina Braun is at the Institute for Neurobiolosy, Brenneckestr. 6, 0-3090 Magdeburg, FRG.
259
which summarizes where they are localized and their possible physiological functions. Not listed in Table I is another protein family called the annexins members of which interact with phospholipids in a Ca2+-dependent manner 8'9. They are also present in neuronal and non-neuronal cells of the brain 1° but have only recently been examined in relation to neurological diseases 11. One member of this family, annexin I (a phosphoprotein and a major substrate of the epidermal growth factor receptor, previously known as lipocortin), appears to be selectively distributed in glia cells of the human CNS 12. The appearance of immunoreactivity to annexin l in reactive astrocytes and macrophages surrounding lesions indicated an involvement of this protein in CNS inflammation and repair. Interestingly, some of these annexins, which are 'Janusfaced' and also integral membrane proteins a3, were reported to exhibit voltage-dependent Ca 2+ channel activities 14.
Ca2+-binding proteins in epilepsy and ischemia Ca 2+ overload as a result of seizures or ischemia is supposed to activate biochemical processes, leading to enzymatic breakdown of proteins and lipids, malfunctioning of mitochondria, energy failure and ultimately cell death 1S. There is experimental evidence 16 that Fig. 1. Ca2+-binding proteins in human dentate gyms. Parvalbumin (PV) and calretinin (CAR) label electrically induced irreversible different subtypes of interneurons, calbindin-D28K (CaBP) labels granule cells, and Sl OOfl protein depolarization of hippocampal labels glial cells that extend their processes densely over granule and hilar neurons. neurons, which may be an early indication of neuronal damage, binding proteins are expressed in a cell-type-specific could be prevented by injecting a Ca2+ chelator and fashion. A central question concerns their biological thereby increasing intracellular Ca 2+ buffering cafunctions and their mechanism of action and interac- pacity. Thus, it is reasonable to assume that neurons tion. There is increasing evidence that these proteins containing certain intracellular Ca2+-binding proteins, not only bind and transport Ca 2+ but that they also and therefore having a greater capacity to buffer Ca 2+, have additional functions that may be independent of would be more resistant to degeneration. However, this ion (e. g. calmodulin is required for growth but can investigations of the vulnerability of such neurons in perform this function without binding Ca2+) 4. It is also the human brain as well as in experimental animal possible that some members of this protein family are models have revealed contradictory results concernsecreted and act extracellularly. Two such members ing the postulated protective role of Ca2+-binding are S10013, which acts as a neurite extension factor s, proteins in seizures and ischemia. and [~-parvalbumin (also named avian thymic horThe increase in intracellular levels of Ca2+ that mone), which promotes immunological maturation of occurs as a result of excitatory amino acid receptor chicken bone marrow cells in culture 6'7. Presently, activation has been suggested to be the initiating Ca2+-binding proteins such as parvalbumin, calbindin factor in seizure-associated degeneration and neurand calretinin are extensively used as neuronal onal death 17. Since only certain subsets of neurons are markers, since these soluble proteins fill the cyto- susceptible to irreversible damage, the positive correplasm of neuronal processes and so facilitate studies lation between the level of parvalbumin or calbindinof neuronal shape, connectivity and functional special- D28K in certain hippocampal neuron populations and ization (see Fig. 1). Those members of this protein their relative resistance to seizure-induced neuronal family that occur in the brain are listed in Table I, damage supports a neuroprotective function for Ca 2+260
TIN& Vol. 15, No. 7, 1992
TABLE I. Ca2+-binding proteins in the brain Proteins with an EF-hand structural motif
Localization
Suggested functions
Neurodegenerative disorders References associated with abnormal protein
Calmodulin
Ubiquitous
Mediates many Ca2+dependent processes
Alzheimer's disease
39
Parvalbumin
Neurons
Ca 2+
buffering and transport, protective role in Ca 2+ overload
Alzheimer's d i s e a s e , epilepsy, ischemia, Pick's disease, Down syndrome, meningiomas, neurofibromatosis
35-38,42,43 20,21,23,63 33 47 57 75
Calbindin-D28K
Neurons (expression regulated by corticosterone in hippocampus)
Ca2+ buffering and transport, protective role in Ca 2+ overload
Alzheimer's disease, epilepsy, ischemia, Parkinson's disease, Down syndrome
40,64 21,24,26 33 49 57
Calretinin
Neurons
Ca 2+
?
32,65,66
Calcineurin
Neurons
Calmodulin-dependent phosphatase, target of cyclophilin-cyclosporin complexes
?
67,68
Calpain
Neurons, astroglia and microglia
Ca2+-activated protease
Alzheimer's disease, ischemia 36,69,70
SlOOc~
Neurons
$10015
Glia cells
Involved in growth and differentiation, assembly and disassemblyof microtubules and actin filaments, phosphorylation, neurite extension (extracellular function)
Alzheimer's disease, 45,51,54 Down syndrome, acquired immune deficiency syndrome (AIDS)
Recoverin or visinin, frequinin, p26
Photoreceptor layer in retina
Phototransduction, activates guanylate cyclase to restore dark state
Retinopathy
buffering and transport, phosphorylation
?
binding proteins 18'19. However, while some authors report that in human epileptic brain tissue the neurons in the hippocampus that are immunoreactive to parvalbumin and calbindin-D28K are relatively spared from degeneration and only some granule cells that stain positively for calbindin-D28K are lost TM, other authors report a clear loss of neurons immunoreactive to parvalbumin and calbindin-D28K (Ref. 21). One of the widely used experimental models for epilepsy is the 'kindling' stimulation. The term 'kindling' refers to a phenomenon in which repeated administration of an initially subconvulsive electrical stimulus results in progressive intensification of seizure activity, culminating in a generalized seizure. This effect is long lasting, since animals left unstimulated for as long as 12 months after kindling will respond to electrical stimuli with a seizure. Focal stimulation of the perforant path to the hippocampus leads to a reduction only in a subset of GABAergic dentate basket cells, which are immunoreactive to parvalbumin, but produces no changes in calbindin-D28K immunoreactivity in granule cells ')2. If, for example, kindling stimulation is applied to the commissural fibers, the number of parvalbuminimmunoreactive neurons increases z3. In neither of these stimulation paradigms is it clear whether changes in parvalbumin immunoreactivity reflect an TIN& Vol. 15, No. 7, 1992
58
72-74
actual numerical increase or decrease in the respective neurons, or whether they reflect changes in the intracellular concentration of parvalbumin, or conformational changes of this protein, which as a result is no longer recognized by the appropriate antibody. Regardless of which theory is correct, these changes in parvalbumin immunoreactivity could indicate functional alterations in the respective neuron population; for instance, in terms of neuronal activity. Assuming that parvalbumin indicates a high level of neuronal activity, the reduction in parvalbumin content or in the number of parvalbumin-immunoreactive neurons in the dentate gyrus could explain the reduced GABAergic inhibition in this area 22. However, other areas in the same preparation that displayed a decrease in inhibition, such as the CA1 hippocampal area, did not show any change in parvalbumin immunostaining. Similarly puzzling are the reported changes in calbindin-D28K levels following neuronal stimulation. While stimulation of the perforant path leads to a transient increase in the level of calbindin-D28K mRNA24, neither amygdaloid nor commissural kindling affects calbindin-D28K levels, although both decrease the calbindin-D28K immunoreactivity in dentate granule cells - including mossy fibers'25 "26". These experiments demonstrate that neuronal 261
activation can modulate the concentration of calbindin-D28K as well as the expression of mRNA encoding the protein. It remains unclear whether the increase in mRNA levels is the result of increased transcription of the calbindin-D28K gene, altered mRNA turnover, or both. Since the authors who reported increased mRNA levels did not check whether the protein itself is also increased (which would be contradictory to the findings of other authors) 25, the functional link between mRNA and protein levels remains unclear. The mechanism by which calbindin-D28K buffers intracellular Ca2+ may be a dual one. K6hr and colleagues 27 recently demonstrated a markedly enhanced Ca2+-dependent inactivation of Ca2+ currents activated by high voltage in kindled granule cells with reduced calbindin-D28K levels, and Morgan et al.28 provided evidence that calbindin-D28K activates Ca2+-Mg2+ ATPase in the plasma membrane. The observed contradictory changes in the levels of parvalbumin and calbindin-D28K may be explained by the different stimulation paradigms, as well as by the different time windows during which the effects of stimulation were analysed [short-term (15 min) versus long-term (several hours or days)]. Furthermore, the stimulation of different afferent pathways excites different neuronal subpopulations, the intrinsic connectivities of which (together with different transmitter sensitivities) may determine their vulnerability to degeneration. Since both commissural and perforant paths are glutamatergic and since it is a general assumption that neuronal degeneration is mediated by excitotoxic amino acids, the differential sensitivity of neurons containing Ca2+-binding proteins to these transmitters is important. Parvalbumin-immunoreactive neurons were found to be selectively vulnerable in vitro to the non-NMDA agonists kainate and a-amino3-hydroxy-5-methyl-4-isoxazole propionate [AMPA], but not to NMDA2". Similar results were reported for striatal parvalbumin-immunoreactive neurons 3° in vivo. Cultured hippocampal neurons immunoreactive to calbindin-D28K seem to be relatively resistant to glutamate-induced neurotoxicity31. Calretinin is a Ca2+-binding protein expressed mainly in nerve cells. Although it shows amino acid sequence similarity to calbindin-D28K, the two proteins are expressed in largely separate sets of neurons. Calretinin-immunoreactive neurons, which form a separate subpopulation of hippocampal interneurons (Fig. 1), can tolerate toxic concentrations of glutamate, NMDA, kainate and quisqualate in vitro 3'2. This selective sensitivity of neurons, characterized by their parvalbumin, calbindin-D28K or calretinin content, to specific glutamate receptor systems does not yet allow a conclusive interpretation of the various effects summarized above. The striking similarity between st~ctural damage in conditions of epilepsy and in ischemia has led to the hypothesis that epileptic brain damage occurs as a result of cellular hypoxia 15. Since hypoxic cells have to cope with abnormally high intracellular Ca2+ concentrations, it is tempting to speculate that those cells equipped with additional Ca2+ buffers in the form of Ca'~+-binding proteins are less vulnerable. Again, experimental data are inconsistent. For instance, after transient ischemia, the number and staining 262
intensity of parvalbumin-immunoreacfive somata and fibers of the CA1 and CA3 hippocampal areas and the hilus transiently decrease, whereas in terminals parvalbumin immunoreactivity remains unchanged33. Within days, parvalbumin immunoreactivity reappears, first in somata and then in fibers. This points to a differential regulation of the protein within cellular compartments. In contrast33, calbindin-D28K immunoreactivity in CA1 neurons is permanently lost, whereas calbindin-D28K immunoreactivity in interneurons and dentate granule cells remains almost unaffected. Although these neurons seem to be able to regulate the levels of intracellular CaZ+-binding proteins in response to altered blood and oxygen supply, studies of various brain areas using combined immunocytochemical and silver-staining techniques to follow degeneration after transient ischemia did not reveal any consistent or systematic relationship between neuronal calbindin-D28K or parvalbumin content and vulnerability:~4.
Ca2÷-binding proteins in chronic neurodegenerative diseases (i) Alzheimer's disease. Results from studies on human pathologic material are inconsistent. For instance, whereas some authors reported no changes in the number of parvalbumin-immunoreactive neurons in prefrontal, frontal and inferior temporal cortex3s':~6, others found a decrease in the number and cell size of parvalbumin-immunoreactive neurons in temporal, parahippocampal, parietal and frontal cortex 37"3s. An overall decrease in the levels of calbindin-D28K, measured by radioimmunoassay and immunocytochemistry, has been found in the temporal, parietal and frontal cortex of brains of patients with Alzheimer's disease3~'4°. However, other authors report unchanged calbindin-D28K mRNA and protein levels in cortical areas, but instead a dramatic decrease in the striatum, hippocampus and nucleus raph~ dorsalis '11. In the hippocampus, parvalbumin-immunoreactive neurons are lost only in certain subfields such as CA3, the subiculum and presubiculum 42. In the prefrontal cortex only the interneurons in layers V and VI and in pyramidal cells in the deep layer III that stain positively for calbindin-D28K are reduced in number, whereas the interneurons immunoreactive for calbindin-D28K in layer II and upper layer III are unaffected43. Again, it seems that neurons that contain a certain Ca2+-binding protein are not necessarily predisposed to react in similar ways. Calpain immunoreactivity is present in cells undergoing tangle formation; such neurons are reduced over the course of Alzheimer's disease44. The ubiquitous protein calmodulin is reduced in the cortical areas of brains of patients with this disease3"~. S100 protein is increased in reactive astrocytes and microglia in the temporal lobe of patients with Alzheimer's disease 45. In this condition, as well as in Down syndrome, a variety of abnormally phosphorylated microtubule-associated proteins (MAPs) (tau and MAP2, among others) form insoluble complexes called paired helical filaments. These filaments are the primary constituents of neurofibrillary tangles. Senile plaques are amorphous extracellular structures that are composed of amyloid and other proteins, possibly TINS, Vol. 15, No. 7, 1992
including cytoskeletal-associated components. S10013 modulates the phosphorylation of at least one MAP (tan) involved in these pathological processes and is overexpressed in both conditions, suggesting that it may be involved in these disorders. Although the etiological role of S100[~ in Alzheimer's disease has not yet been proven, transgenic mice that express abnormal levels of S10013 have recently been shown to have significant behavioral and cognitive abnormalities and may serve as one experimental model for Alzheimer's disease 46. Some of the contradictory findings concerning Ca"+-binding proteins in Alzheimer's disease - especially in human material - might be due to different experimental protocols, such as the use of different antibodies and fLxation procedures, different postmortem intervals, and the variability in the ages and clinical histories of the patients studied. Furthermore, pathologically altered afferent and intrinsic connectivities and transmitter sensitivities may account for the observed region-specific variability. (2) Other neurodegenerative diseases. In Pick's disease, a single study reported no changes in parvalbumin-immunoreactive neurons in frontal and temporal cortex 47. However, other workers report a decrease of parvalbumin-immunoreactive neurons in temporal, parahippocampal, parietal and cerebellar cortex 48, which is comparable to the effects observed in brains of patients with Alzheimer's disease and supranuclear palsy. In Parkinson's disease a dramatic reduction in calbindin-D28K mRNA and protein levels is found in the substantia nigra, hippocampus and the nucleus raph6 dorsalis 41, but the pigmented neurons that are immunoreactive to calbindin-D28K in the substantia nigra of these patients seem to be selectively spared from degeneration 4~. In the brains of patients with acquired immune deficiency syndrome (AIDS), the number and size of astrocytes expressing S100/3 protein are greatly increased 5°. (3) Down syndrome (trisomy 21). Since the gene encoding the glial $10013 protein is located on chromosome 21 (Ref. 51), it is not surprising that metabolism of S100 is altered in patients with trisomy 21. Since levels of $100 increase in the fetal brain during maturation ~2, and since they can be regulated by nerve growth factor ~a, impaired regulation of this protein may result in maldevelopment. An elevated level of $10013 in the lymphocyte fraction and blood of patients with Down syndrome has been reported, whereas levels of neuronal $100~ are normal in such patients 54. Since elevation of $100 protein in cerebrospinal fluid seems to be a relatively sensitive indicator of brain damage 55, increased levels of this protein could reflect glia-associated degenerative changes in these patients. Furthermore, $100[3 (but not S100o:) regulates intracellular Ca 2+ concentrations in glia and neurons both dependently and independently of extracellular Ca '~+ (Ref. 56). Thus, elevated $10013 levels may induce CaZ+-regulated neuronal degeneration. In view of the well-known rapid ageing of Down syndrome patients, it should be mentioned that brains of patients with Alzheimer's disease also show elevated $100 levels45. Neurons immunoreactive to parvalbumin and calbindin-D28K are reduced in the cortex of the brains of patients with Down syndrome 57 where they form two separate subpopulations of interneurons. TINS, Vol. 15, No. 7, 1992
Ca2+-binding proteins as diagnostic tools As already mentioned above, Ca2+-binding proteins such as $10058 have also been measured in the cerebrospinal fluid and the blood of patients with cerebral infarction, transient ischemic attack, hemorrhage, and head injury 59. A rise in S10013 levels was also found in the blood of patients with Down syndrome 49. In the future these and other Ca2+binding proteins might be used as selective markers to estimate the extent of brain damage in various neurological disorders and also to classify various brain tumors in children 6° and in adults 61.
Concluding remarks Studies of aberrations in the second messenger functions of Ca 2+, which are partially reflected by changed levels of some intraceUular Ca~+-binding proteins, may help to elucidate some of the complex neurological disturbances found in neurodegenerative and bipolar affective disorders. Minute changes in intracellular Ca2+ have profound effects on neuronal cell functions, and future studies should therefore focus on the physiological roles of these proteins in the CNS, using transgenic modeling of neurodegenerative events 6" and transfection of neuronal cell lines with cDNAs encoding Ca~+-binding proteins. The outcome of these results may then also be relevant to medication developed to correct hyperactivity of intracellular Ca 2÷. Selected references 1 Dubovsky, S. L., Lee, C., Christiano, J. and Murphy, J. (1991) Lithium 2, 167-174 2 Kretsinger, R. H., Tolbert, D., Nakayama, S. and Pearson, W. (1991 ) in Novel Calcium-Binding Proteins: Fundamentalsand Clinical Implications (Heizmann, C. W., ed.), pp. 17-37, Springer-Verlag 3 Heizmann, C. W. and Hunziker, W. (1991) Trends Biochem. Sci. 16, 98-103 4 Geiser, J. R., van Tuinen, D., Brockerhoff, S. E., Neff, M. M. and Davis, T. N. (1991) Ceil 65, 949-959 5 Kligman, D. and Marshak, D. R. (1985) Proc. NatlAcad. Sci. USA 82, 7136--7139 6 Brewer, J. M., Wunderlich, J. K. and Ragland, W. (1990) Biochimie 72,653-660 7 Kuster, T., Staudenmann, W., Hughes, G. J. and Heizmann, C. W. (1991) Biochemistry 30, 8812-8816 8 Crumpton, M. J. and Dedman, J. R. (1990) Nature 345, 212 9 Moss, S. E., Edwards, H. E. and Crumpton, M. J. (1991) in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ed.), pp. 535-566, SpringerVerlag 10 Woolgar, J. A., Boustead, C. M. and Walker, J. H. (1990) J. Neurochem. 54, 62-71 11 ReRon, J. K. etal. (1991) J. Exp. Med. 174, 305-310 12 Johnson, M. D., Kamso-Pratt, J. M., Whetsell, W. O. and Pepinsky, R. B. (1989) Am. J. Clin. Pathol. 92,424-429 13 Huber, R. etal. (1992)J. Mol. Biol. 223,683-704 14 Burns, A. L. eta/. (1989) Proc. Natl Acad. Sci. USA 86, 3798-3802 15 Siesj6, B. K. and Wieloch, T. (1986) in Advances in Neurology (Delgado-Escueta, A. V. and Ward, A. A., Jr, eds), pp. 813-847, Raven Press 16 Scharfman, H. E. and Schwartzkroin, P. A. (1989) Science 246, 257-260 17 Meldrum, B. S. (1981) in Metabolic Disorders of the Nervous System (Rose, F. C., ed.), pp. 175-187, Pitman 18 Sloviter, R. S. (1989) J. Neurol. Neurosurg. Psychiatry 45, 1130-1135 19 Leranth, C. and Ribak, C. E. (1991) Exp. Brain Res. 85, 129-136 20 Sloviter, R. S., Solias, A. L., Barbaro, N. M. and Laxer, K. D. (1991) J. Comp. NeuroL 308, 381-396 21 Vonau, M. and T&k, I. (1991) Soc. Neurosci. Abstr. 17, 1260 22 Sloviter, R. S. (1991) Hippocampus 1, 41-66 263
Acknowledgements We apologizeto many colleagues whoseimportant work cou/dnot be dted due to space limitations. We would like to thank A. Rowlersonfor helping with the preparation of this manuscript, M. K#lenfor typing it and K. Wehnerfor photographicalwork. Thisstudy was supportedin part by the SwissNational ScienceFoundation (to C W.H., grant no. 31--30742.91), Hartmann-Miiller Stiftung f6r Medizimsche Forschung, Ciba-
Gegy Jubilaumsstiftung, the Deutsche Bundesministerium f~r Forschungund Technologie,and the Deutsche Forschungsgemeinschaft (to K.B., grant no. Br950/4-5).
23 Kamphuis, W., Huisman, E., Wadman, W. J., Heizmann, C. W. and Lopes da Silva, F. H. (1989) Brain Res. 479, 23-34 24 Lowenstein, D. H., Miles, M. F., Hatam, F. and McCabe, T. (1991) Neuron 6, 627-633 25 Baimbridge, K. G., Mody, I. and Miller, J. J. (1985) Epilepsia 26, 460-465 26 Sonnenberg, J. L. etal. (1991) Mol. Brain Res. 9, 179-190 27 K6hr, G., Lambert, C. E. and Mody, I. (1991) Exp. Brain Res. 85, 543-551 28 Morgan, D. W., Welton, A. F., Heick, A. E. and Christakos, S. (1986) Biochem. Biophys. Res. Commun. 138, 547-553 29 Weiss, J. H., Koh, J. Y., Baimbridge, K. G. and Choi, D. W. (1990) Neurology 40, 1288-1292 30 Waldvogel, H. J., Faull, R. L. M., Williams, M. N. and Dragunow, M. (1991) Brain Res. 546, 329-335 31 Baimbridge, K. G. and Kuo, J. (1988) Soc. Neurosci. Abstr. 14, 1264 32 Winsky, L., Jacobowitz, D. M., Kispert, J. and Henneberry, R. C. (1991) in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ed.), pp. 277-300, Springer-Verlag 33 Johansen, F. F., Tonder, N., Zimmer, J., Baimbridge, K. G. and Diemer, N. H. (1990) Neurosci. Left. 120, 171-174 34 Freund, T. F., Buszaki, G., Leon, A., Baimbridge, K. G. and Somogyi, P. (1990) Exp. Brain Res. 83, 55-66 35 Hof, P. R. et aL (1991)J. NeuropathoL Exp. Neurol. 50, 451-462 36 Iwamoto, N. and Emson, P. C. (1991) Neurosci. Lett. 128, 81-84 37 Arai, H., Emson, P. C., Mountjoy, C. Q., Carassco, L. H. and Heizmann, C. W. (1987) Brain Res. 418, 164-169 38 Satoh, J., Tabira, T., Sano, M,, Nakayama, H. and Tateishi, J. (1991) Acta Neuropathol. 81,388-395 39 McLacHan, D. R. C., Wong, L., Bergeron, C. and Baimbridge, K. G. (1987)Alzheimer Dis. and Assoc. Disord. 1, 171-179 40 Ichimiya, Y., Emson, P. C., Mountjoy, C. Q., Lawson, E. E. M. and Heizmann, C. W. (1988) Brain Res. 475, 156-159 41 lacopino, A. M. and Christakos, S. (1990)J. Biol. Chem. 265, 10177-10180 42 Brady, D. R. and Mufson, E. J. (1991) Soc. Neurosci. Abstr. 17, 691 43 Hof, P. R. and Mordson, J, H. (1991) Exp. Neurol. 111, 293-301 44 Iwamoto, N., Thangnipon, W., Crawford, C. and Emson, P. C. (1991) Brain Res. 561, 177-180 45 Griffin, W. S. eta/. (1989) Proc. Natl Acad. Sci. USA 86, 7611-7615 46 Reeves, R., Crowley, M., Hilt, D. C., Moran, T. and Gerhard, J. (1990) Mouse Molec. Genetics Meeting, Cold Spring Harbor 47 Arai, H., Noguchi, I., Makino, Y., Kosaka, K. and Heizmann,
C. W. (1991) J. Neurol. 238, 200-202 48 Satoh, J., Tabira, T., Sano, M., Nakayama, H. and Tateishi, J. (1991) Acta NeuropathoL 81, 388-395 49 Yamada, T., McGeer, P. L., Baimbridge, K. G. and McGeer, E. G. (1990) Brain Res. 526, 305--307 50 Griffin, W. S. T., Stanley, L. C., Mrak, R. E. and Perrot, L. J. (1991) Soc. Neurosci. Abstr. 17, 1273 51 AIIore, R. et al. (1988)Science 239, 1311-1313 52 Zuckerman, J. E., Herschman, H. R. and Levine, L. (1970) J. Neurochem. 17, 247-251 53 Zimmer, D. B., Cochran, K. H. and Strada, S. J. (1991) Soc. Neurosci. Abstr. 17, 1314 54 Kato, K. eta/. (1990)J. Mol. Neurosci. 2, 109-113 55 Sindic, C. J. M., Chalon, M. P., Cambiaso, C. L., Laterre, E. C. and Masson, P. L. (1982)J. Neurol. Neurosurg. Psychiatry 45, 1130-1135 56 Barger, S. W. and van Eldik, L. J. (1991) Soc. Neurosci. Abstr. 17, 1500 57 Kobayashi, K. et al. (1990) Neurosci. Lett. 113, 17-22 58 Hilt, D. C. and Kligman, D. (1991)in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ecl.), pp. 65-103, Springer-Vedag 59 Persson, L. etal. (1987) Stroke 19, 911-918 60 Ishiguro, Y., Kato, K., Ito, T. and Nagaya, M. (1983) Cancer Res. 43, 6080-6084 61 Fagnart, O. C., Sindic, C. J. M. and Laterre, C. (1988) Clin. Chem. 34, 1387-1391 62 Sofroniew, M. V. and Staley, K. (1991) Trends Neurosci. 14, 513-514 63 Sloviter, R. S. (1987) Science 235, 73-76 64 lacapino, A. M. and Christakos, S. (1990) Proc. Natl Acad. Sci. USA 87, 4078-4082 65 Rogers, J. H. (1991) in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ed.), pp. 251-276, Springer-Verlag 66 Winsky, L. and Jacobowitz, D. M. (1991) Neurosci. Lett. 131, 79-82 67 Goto, S. eta/. (1988) Exp. Brain Res. 69, 645-650 68 Liu, J. etal. (1991) Cell 66, 807-815 69 Perlmutter, L. S., Gall, C., Baudry, M. and Lynch, G. (1990) J. Comp. Neurol. 296, 269-276 70 Nilson, E., Ostwald, K. and Karlsson, J. O. (1991) Mol. Chem. Neuropathol. 14, 99-105 71 Selinfreund, R. H., Barger, S. W., Pledger, W. J. and van Eldik, L. J. (1991) Proc. NatlAcad. Sci. USA 88, 3554-3558 72 Stryer, L. (1991)J. Biol. Chem. 266, 10711-10714 73 Lambrecht, H-G. and Kock, K-W. (1991) EMBO J. 10, 793-798 74 Polans, A. S., Buczylko, J., Crabb, J. and Palczewski, K (1991) J. Cell. Biol. 112, 981-989 75 Ritzler, J. M. et a/. (1992) Genomics 12, 567-572
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TIN& Vol. 15, No. 7, 1992
21 22 23 24 25 26
242,427-432 28 Furness, D. N. and Hackney, C. M. (1985) Hear. Res. 18, 177-188 29 Osborne, M. P., Comis, S. D. and Pickles, J. O. (1988) Hear. Res. 35, 99-108 30 Neugebauer, D-Ch. and Thurm, U. (1987) Cell Tissue Res. 249, 199-207 31 Pickles, J. O., Rouse, G. W. and von Perger, M. (1991) Scanning Microsc. 5, 111 5-1128 32 Comis, S. D., Pickles, J. O. and Osborne, M. P. (1985) J. Neurocytol. 14, 113-130 33 Rhys-Evans, P. H., Comis, S. D., Osborne, M. P., Pickles, J. O. and Jeffries, D. J. R. (1985) J. Laryngol. Otol. 99, 11-19 34 Rouse, G. W. and Pickles, J. O. (1991) J. NlorphoL 209,
111-120 35 Szabo, T. (1974) in Handbook of Sensory Physiology 3 (Fessard, A., ed.), pp. 13-58, Springer 36 Assad, J. A. and Corey, D. P. J. Neurosci. (in press) 37 Eatock, R. A., Corey, D. P. and Hudspeth, A. J. (1987) J. Neurosci. 7, 2821-2836 38 Howard, J. and Hudspeth, A. J. (1987) Proc. NatlAcad. Sci. USA 84, 3064-3068 39 Shepherd, G. M. G. et al. J. Gen. Physiol. (in press) 40 Stevens, S. S. and Davis, H. (1983) Hearing: Its Psychology and Physiology, Wiley 41 Hillman, D. E. (1969) Brain Res. 13, 407-412 42 Dallos, P. (1973) The Auditory Periphery, Academic Press 43 Hackney, C. M., Furness, D. N. and Benos, D. J. (1991) Scanning Microsc. 5, 741-746 44 Jorgensen, F. and Ohmori, H. (1988) J. Physiol. 403, 577-588 45 Shepherd, G. M. G., Barres, B. A. and Corey, D. P. (1989) Proc. Natl Acad. Sci. USA 86, 4973-4977 46 Gillespie, P. G. and Hudspeth, A. J. (1991)J. Cell Biol. 112, 625-640 47 Shepherd, G. M. G., Corey, D. P. and Block, S. M. (1990) Proc. Natl Acad. Sci. USA 87, 8627-8631
Acknowledgements Work in the authors' laboratories was supported by the MRC of the UK and the Australian ResearchCouncil (J.O.P.)and by the Nab'onallnstitutes of Health, the Office of Naval Research, and the Howard Hughes Medical Institute (D.P.C.).
Changesin Caz +-bindingproteinsin human neurodepenerativedisorders Claus W. Heizmann
The cellular distribution of Cae+-binding proteins has been extensively studied during the past decade. These proteins have proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major roles are assumed to be Ca e+ buffering and transport, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca e+ homeostasis, a protective role for cae+-binding proteins in certain neuron populations has been postulated. As massive neuronal degeneration takes place in several brain diseases of humans, such as Alzheimer's disease, Parkinson's disease and epilepsy, changes in the expression of Ca 2+binding proteins have therefore been studied during the course of these diseases. Although the data from these studies are inconsistent, the detection and quantification of Cae+-binding proteins and the neuron populations in which they occur may nevertheless be useful to estimate, for example, the location and extent of brain damage in the various neurological disorders. I f future studies advance our knowledge about the physiological functions of these proteins, the neuronal systems in which they are expressed may become important therapeutical targets for preventing neuronal death in an array of neurodegenerative diseases. More than 7000 articles on calcium were published in 1991 (a figure found using the Medline database, with 'calcium' as the search word). This emphasizes the tremendous interest and progress in Ca2+-related research. In nerve cells, Ca 2+ ions activate and regulate a number of key processes, including fast axonal transport of substances, synthesis and release of some neurotransmitters, and membrane excitability. It is also thought that in the CA1 hippocampal region of the brain, Caz+ might induce long-term potentiation and memory storage mechanisms. The Ca2+ message is converted into an intracellular response - in many cases by CaZ+-binding proteins TINS, Vol. 15, No. 7, 1992
and Katharina Braun
that are involved in a wide variety of activities, such as cytoskeletal organization, cell motility and differentiation, cell-cycle regulation, and Ca2+ buffeting and transport. It might therefore be possible that altered levels of some Ca2+-binding proteins (e.g. due to deletion or mutation of the corresponding genes) could lead to an impaired Ca2+ homeostasis in nerve cells and to pathological conditions. Using mostly immunohistochemical techniques, several research groups have now started to search for altered expression of the CaZ+-binding proteins parvalbumin, calbindin-D28K and S100 in affected brain regions of patients suffering from acute insults, such as stroke and epileptic seizures, and from chronic neurodegenerative disorders, such as Alzheimer's, Huntington's, Parkinson's and Pick's diseases. In addition, altered Ca2+ levels have been found in platelets of patients with bipolar affective disorders 1, and Ca 2+ antagonists have been suggested for treatment of psychotic depression. The few proteins that have been investigated in these pathological states belong to a large family of more than 200 members. These proteins are characterized by a common structural motif, the EF-hand, which is present in multiple copies and binds Ca2+ selectively and with high affinity. Each of these domains consists of a loop of 12 amino acids that is flanked by two ochelices. This structural principle was first identified as a result of studying the crystal structure of the Ca2+-binding protein parvalbumin isolated from carp, and is designated the EF-hand because of the arrangement of the E and F helices of parvalbumin. A consensus amino acid sequence for this motif has aided the identification of many new members of this protein family2'3. Ca2+-binding proteins with known functions, such as calmodulin, troponin C, myosin light chains, calpain, calcineurin and recoverin, are far outnumbered by those with unidentified roles. Most Ca2+-
© 1992, ElsevierSciencePublishersLtd,(UK)
Claus W. Heizrnannis at the Dept of Pediatrics, Division of Clinical Chemistry, Universityof Zurich, 5teinwiesstr. 75, CH-8032 Zurich, Switzerland,and
Katharina Braun is at the Institute for Neurobiolosy, Brenneckestr. 6, 0-3090 Magdeburg, FRG.
259
which summarizes where they are localized and their possible physiological functions. Not listed in Table I is another protein family called the annexins members of which interact with phospholipids in a Ca2+-dependent manner 8'9. They are also present in neuronal and non-neuronal cells of the brain 1° but have only recently been examined in relation to neurological diseases 11. One member of this family, annexin I (a phosphoprotein and a major substrate of the epidermal growth factor receptor, previously known as lipocortin), appears to be selectively distributed in glia cells of the human CNS 12. The appearance of immunoreactivity to annexin l in reactive astrocytes and macrophages surrounding lesions indicated an involvement of this protein in CNS inflammation and repair. Interestingly, some of these annexins, which are 'Janusfaced' and also integral membrane proteins a3, were reported to exhibit voltage-dependent Ca 2+ channel activities 14.
Ca2+-binding proteins in epilepsy and ischemia Ca 2+ overload as a result of seizures or ischemia is supposed to activate biochemical processes, leading to enzymatic breakdown of proteins and lipids, malfunctioning of mitochondria, energy failure and ultimately cell death 1S. There is experimental evidence 16 that Fig. 1. Ca2+-binding proteins in human dentate gyms. Parvalbumin (PV) and calretinin (CAR) label electrically induced irreversible different subtypes of interneurons, calbindin-D28K (CaBP) labels granule cells, and Sl OOfl protein depolarization of hippocampal labels glial cells that extend their processes densely over granule and hilar neurons. neurons, which may be an early indication of neuronal damage, binding proteins are expressed in a cell-type-specific could be prevented by injecting a Ca2+ chelator and fashion. A central question concerns their biological thereby increasing intracellular Ca 2+ buffering cafunctions and their mechanism of action and interac- pacity. Thus, it is reasonable to assume that neurons tion. There is increasing evidence that these proteins containing certain intracellular Ca2+-binding proteins, not only bind and transport Ca 2+ but that they also and therefore having a greater capacity to buffer Ca 2+, have additional functions that may be independent of would be more resistant to degeneration. However, this ion (e. g. calmodulin is required for growth but can investigations of the vulnerability of such neurons in perform this function without binding Ca2+) 4. It is also the human brain as well as in experimental animal possible that some members of this protein family are models have revealed contradictory results concernsecreted and act extracellularly. Two such members ing the postulated protective role of Ca2+-binding are S10013, which acts as a neurite extension factor s, proteins in seizures and ischemia. and [~-parvalbumin (also named avian thymic horThe increase in intracellular levels of Ca2+ that mone), which promotes immunological maturation of occurs as a result of excitatory amino acid receptor chicken bone marrow cells in culture 6'7. Presently, activation has been suggested to be the initiating Ca2+-binding proteins such as parvalbumin, calbindin factor in seizure-associated degeneration and neurand calretinin are extensively used as neuronal onal death 17. Since only certain subsets of neurons are markers, since these soluble proteins fill the cyto- susceptible to irreversible damage, the positive correplasm of neuronal processes and so facilitate studies lation between the level of parvalbumin or calbindinof neuronal shape, connectivity and functional special- D28K in certain hippocampal neuron populations and ization (see Fig. 1). Those members of this protein their relative resistance to seizure-induced neuronal family that occur in the brain are listed in Table I, damage supports a neuroprotective function for Ca 2+260
TIN& Vol. 15, No. 7, 1992
TABLE I. Ca2+-binding proteins in the brain Proteins with an EF-hand structural motif
Localization
Suggested functions
Neurodegenerative disorders References associated with abnormal protein
Calmodulin
Ubiquitous
Mediates many Ca2+dependent processes
Alzheimer's disease
39
Parvalbumin
Neurons
Ca 2+
buffering and transport, protective role in Ca 2+ overload
Alzheimer's d i s e a s e , epilepsy, ischemia, Pick's disease, Down syndrome, meningiomas, neurofibromatosis
35-38,42,43 20,21,23,63 33 47 57 75
Calbindin-D28K
Neurons (expression regulated by corticosterone in hippocampus)
Ca2+ buffering and transport, protective role in Ca 2+ overload
Alzheimer's disease, epilepsy, ischemia, Parkinson's disease, Down syndrome
40,64 21,24,26 33 49 57
Calretinin
Neurons
Ca 2+
?
32,65,66
Calcineurin
Neurons
Calmodulin-dependent phosphatase, target of cyclophilin-cyclosporin complexes
?
67,68
Calpain
Neurons, astroglia and microglia
Ca2+-activated protease
Alzheimer's disease, ischemia 36,69,70
SlOOc~
Neurons
$10015
Glia cells
Involved in growth and differentiation, assembly and disassemblyof microtubules and actin filaments, phosphorylation, neurite extension (extracellular function)
Alzheimer's disease, 45,51,54 Down syndrome, acquired immune deficiency syndrome (AIDS)
Recoverin or visinin, frequinin, p26
Photoreceptor layer in retina
Phototransduction, activates guanylate cyclase to restore dark state
Retinopathy
buffering and transport, phosphorylation
?
binding proteins 18'19. However, while some authors report that in human epileptic brain tissue the neurons in the hippocampus that are immunoreactive to parvalbumin and calbindin-D28K are relatively spared from degeneration and only some granule cells that stain positively for calbindin-D28K are lost TM, other authors report a clear loss of neurons immunoreactive to parvalbumin and calbindin-D28K (Ref. 21). One of the widely used experimental models for epilepsy is the 'kindling' stimulation. The term 'kindling' refers to a phenomenon in which repeated administration of an initially subconvulsive electrical stimulus results in progressive intensification of seizure activity, culminating in a generalized seizure. This effect is long lasting, since animals left unstimulated for as long as 12 months after kindling will respond to electrical stimuli with a seizure. Focal stimulation of the perforant path to the hippocampus leads to a reduction only in a subset of GABAergic dentate basket cells, which are immunoreactive to parvalbumin, but produces no changes in calbindin-D28K immunoreactivity in granule cells ')2. If, for example, kindling stimulation is applied to the commissural fibers, the number of parvalbuminimmunoreactive neurons increases z3. In neither of these stimulation paradigms is it clear whether changes in parvalbumin immunoreactivity reflect an TIN& Vol. 15, No. 7, 1992
58
72-74
actual numerical increase or decrease in the respective neurons, or whether they reflect changes in the intracellular concentration of parvalbumin, or conformational changes of this protein, which as a result is no longer recognized by the appropriate antibody. Regardless of which theory is correct, these changes in parvalbumin immunoreactivity could indicate functional alterations in the respective neuron population; for instance, in terms of neuronal activity. Assuming that parvalbumin indicates a high level of neuronal activity, the reduction in parvalbumin content or in the number of parvalbumin-immunoreactive neurons in the dentate gyrus could explain the reduced GABAergic inhibition in this area 22. However, other areas in the same preparation that displayed a decrease in inhibition, such as the CA1 hippocampal area, did not show any change in parvalbumin immunostaining. Similarly puzzling are the reported changes in calbindin-D28K levels following neuronal stimulation. While stimulation of the perforant path leads to a transient increase in the level of calbindin-D28K mRNA24, neither amygdaloid nor commissural kindling affects calbindin-D28K levels, although both decrease the calbindin-D28K immunoreactivity in dentate granule cells - including mossy fibers'25 "26". These experiments demonstrate that neuronal 261
activation can modulate the concentration of calbindin-D28K as well as the expression of mRNA encoding the protein. It remains unclear whether the increase in mRNA levels is the result of increased transcription of the calbindin-D28K gene, altered mRNA turnover, or both. Since the authors who reported increased mRNA levels did not check whether the protein itself is also increased (which would be contradictory to the findings of other authors) 25, the functional link between mRNA and protein levels remains unclear. The mechanism by which calbindin-D28K buffers intracellular Ca2+ may be a dual one. K6hr and colleagues 27 recently demonstrated a markedly enhanced Ca2+-dependent inactivation of Ca2+ currents activated by high voltage in kindled granule cells with reduced calbindin-D28K levels, and Morgan et al.28 provided evidence that calbindin-D28K activates Ca2+-Mg2+ ATPase in the plasma membrane. The observed contradictory changes in the levels of parvalbumin and calbindin-D28K may be explained by the different stimulation paradigms, as well as by the different time windows during which the effects of stimulation were analysed [short-term (15 min) versus long-term (several hours or days)]. Furthermore, the stimulation of different afferent pathways excites different neuronal subpopulations, the intrinsic connectivities of which (together with different transmitter sensitivities) may determine their vulnerability to degeneration. Since both commissural and perforant paths are glutamatergic and since it is a general assumption that neuronal degeneration is mediated by excitotoxic amino acids, the differential sensitivity of neurons containing Ca2+-binding proteins to these transmitters is important. Parvalbumin-immunoreactive neurons were found to be selectively vulnerable in vitro to the non-NMDA agonists kainate and a-amino3-hydroxy-5-methyl-4-isoxazole propionate [AMPA], but not to NMDA2". Similar results were reported for striatal parvalbumin-immunoreactive neurons 3° in vivo. Cultured hippocampal neurons immunoreactive to calbindin-D28K seem to be relatively resistant to glutamate-induced neurotoxicity31. Calretinin is a Ca2+-binding protein expressed mainly in nerve cells. Although it shows amino acid sequence similarity to calbindin-D28K, the two proteins are expressed in largely separate sets of neurons. Calretinin-immunoreactive neurons, which form a separate subpopulation of hippocampal interneurons (Fig. 1), can tolerate toxic concentrations of glutamate, NMDA, kainate and quisqualate in vitro 3'2. This selective sensitivity of neurons, characterized by their parvalbumin, calbindin-D28K or calretinin content, to specific glutamate receptor systems does not yet allow a conclusive interpretation of the various effects summarized above. The striking similarity between st~ctural damage in conditions of epilepsy and in ischemia has led to the hypothesis that epileptic brain damage occurs as a result of cellular hypoxia 15. Since hypoxic cells have to cope with abnormally high intracellular Ca2+ concentrations, it is tempting to speculate that those cells equipped with additional Ca2+ buffers in the form of Ca'~+-binding proteins are less vulnerable. Again, experimental data are inconsistent. For instance, after transient ischemia, the number and staining 262
intensity of parvalbumin-immunoreacfive somata and fibers of the CA1 and CA3 hippocampal areas and the hilus transiently decrease, whereas in terminals parvalbumin immunoreactivity remains unchanged33. Within days, parvalbumin immunoreactivity reappears, first in somata and then in fibers. This points to a differential regulation of the protein within cellular compartments. In contrast33, calbindin-D28K immunoreactivity in CA1 neurons is permanently lost, whereas calbindin-D28K immunoreactivity in interneurons and dentate granule cells remains almost unaffected. Although these neurons seem to be able to regulate the levels of intracellular CaZ+-binding proteins in response to altered blood and oxygen supply, studies of various brain areas using combined immunocytochemical and silver-staining techniques to follow degeneration after transient ischemia did not reveal any consistent or systematic relationship between neuronal calbindin-D28K or parvalbumin content and vulnerability:~4.
Ca2÷-binding proteins in chronic neurodegenerative diseases (i) Alzheimer's disease. Results from studies on human pathologic material are inconsistent. For instance, whereas some authors reported no changes in the number of parvalbumin-immunoreactive neurons in prefrontal, frontal and inferior temporal cortex3s':~6, others found a decrease in the number and cell size of parvalbumin-immunoreactive neurons in temporal, parahippocampal, parietal and frontal cortex 37"3s. An overall decrease in the levels of calbindin-D28K, measured by radioimmunoassay and immunocytochemistry, has been found in the temporal, parietal and frontal cortex of brains of patients with Alzheimer's disease3~'4°. However, other authors report unchanged calbindin-D28K mRNA and protein levels in cortical areas, but instead a dramatic decrease in the striatum, hippocampus and nucleus raph~ dorsalis '11. In the hippocampus, parvalbumin-immunoreactive neurons are lost only in certain subfields such as CA3, the subiculum and presubiculum 42. In the prefrontal cortex only the interneurons in layers V and VI and in pyramidal cells in the deep layer III that stain positively for calbindin-D28K are reduced in number, whereas the interneurons immunoreactive for calbindin-D28K in layer II and upper layer III are unaffected43. Again, it seems that neurons that contain a certain Ca2+-binding protein are not necessarily predisposed to react in similar ways. Calpain immunoreactivity is present in cells undergoing tangle formation; such neurons are reduced over the course of Alzheimer's disease44. The ubiquitous protein calmodulin is reduced in the cortical areas of brains of patients with this disease3"~. S100 protein is increased in reactive astrocytes and microglia in the temporal lobe of patients with Alzheimer's disease 45. In this condition, as well as in Down syndrome, a variety of abnormally phosphorylated microtubule-associated proteins (MAPs) (tau and MAP2, among others) form insoluble complexes called paired helical filaments. These filaments are the primary constituents of neurofibrillary tangles. Senile plaques are amorphous extracellular structures that are composed of amyloid and other proteins, possibly TINS, Vol. 15, No. 7, 1992
including cytoskeletal-associated components. S10013 modulates the phosphorylation of at least one MAP (tan) involved in these pathological processes and is overexpressed in both conditions, suggesting that it may be involved in these disorders. Although the etiological role of S100[~ in Alzheimer's disease has not yet been proven, transgenic mice that express abnormal levels of S10013 have recently been shown to have significant behavioral and cognitive abnormalities and may serve as one experimental model for Alzheimer's disease 46. Some of the contradictory findings concerning Ca"+-binding proteins in Alzheimer's disease - especially in human material - might be due to different experimental protocols, such as the use of different antibodies and fLxation procedures, different postmortem intervals, and the variability in the ages and clinical histories of the patients studied. Furthermore, pathologically altered afferent and intrinsic connectivities and transmitter sensitivities may account for the observed region-specific variability. (2) Other neurodegenerative diseases. In Pick's disease, a single study reported no changes in parvalbumin-immunoreactive neurons in frontal and temporal cortex 47. However, other workers report a decrease of parvalbumin-immunoreactive neurons in temporal, parahippocampal, parietal and cerebellar cortex 48, which is comparable to the effects observed in brains of patients with Alzheimer's disease and supranuclear palsy. In Parkinson's disease a dramatic reduction in calbindin-D28K mRNA and protein levels is found in the substantia nigra, hippocampus and the nucleus raph6 dorsalis 41, but the pigmented neurons that are immunoreactive to calbindin-D28K in the substantia nigra of these patients seem to be selectively spared from degeneration 4~. In the brains of patients with acquired immune deficiency syndrome (AIDS), the number and size of astrocytes expressing S100/3 protein are greatly increased 5°. (3) Down syndrome (trisomy 21). Since the gene encoding the glial $10013 protein is located on chromosome 21 (Ref. 51), it is not surprising that metabolism of S100 is altered in patients with trisomy 21. Since levels of $100 increase in the fetal brain during maturation ~2, and since they can be regulated by nerve growth factor ~a, impaired regulation of this protein may result in maldevelopment. An elevated level of $10013 in the lymphocyte fraction and blood of patients with Down syndrome has been reported, whereas levels of neuronal $100~ are normal in such patients 54. Since elevation of $100 protein in cerebrospinal fluid seems to be a relatively sensitive indicator of brain damage 55, increased levels of this protein could reflect glia-associated degenerative changes in these patients. Furthermore, $100[3 (but not S100o:) regulates intracellular Ca 2+ concentrations in glia and neurons both dependently and independently of extracellular Ca '~+ (Ref. 56). Thus, elevated $10013 levels may induce CaZ+-regulated neuronal degeneration. In view of the well-known rapid ageing of Down syndrome patients, it should be mentioned that brains of patients with Alzheimer's disease also show elevated $100 levels45. Neurons immunoreactive to parvalbumin and calbindin-D28K are reduced in the cortex of the brains of patients with Down syndrome 57 where they form two separate subpopulations of interneurons. TINS, Vol. 15, No. 7, 1992
Ca2+-binding proteins as diagnostic tools As already mentioned above, Ca2+-binding proteins such as $10058 have also been measured in the cerebrospinal fluid and the blood of patients with cerebral infarction, transient ischemic attack, hemorrhage, and head injury 59. A rise in S10013 levels was also found in the blood of patients with Down syndrome 49. In the future these and other Ca2+binding proteins might be used as selective markers to estimate the extent of brain damage in various neurological disorders and also to classify various brain tumors in children 6° and in adults 61.
Concluding remarks Studies of aberrations in the second messenger functions of Ca 2+, which are partially reflected by changed levels of some intraceUular Ca~+-binding proteins, may help to elucidate some of the complex neurological disturbances found in neurodegenerative and bipolar affective disorders. Minute changes in intracellular Ca2+ have profound effects on neuronal cell functions, and future studies should therefore focus on the physiological roles of these proteins in the CNS, using transgenic modeling of neurodegenerative events 6" and transfection of neuronal cell lines with cDNAs encoding Ca~+-binding proteins. The outcome of these results may then also be relevant to medication developed to correct hyperactivity of intracellular Ca 2÷. Selected references 1 Dubovsky, S. L., Lee, C., Christiano, J. and Murphy, J. (1991) Lithium 2, 167-174 2 Kretsinger, R. H., Tolbert, D., Nakayama, S. and Pearson, W. (1991 ) in Novel Calcium-Binding Proteins: Fundamentalsand Clinical Implications (Heizmann, C. W., ed.), pp. 17-37, Springer-Verlag 3 Heizmann, C. W. and Hunziker, W. (1991) Trends Biochem. Sci. 16, 98-103 4 Geiser, J. R., van Tuinen, D., Brockerhoff, S. E., Neff, M. M. and Davis, T. N. (1991) Ceil 65, 949-959 5 Kligman, D. and Marshak, D. R. (1985) Proc. NatlAcad. Sci. USA 82, 7136--7139 6 Brewer, J. M., Wunderlich, J. K. and Ragland, W. (1990) Biochimie 72,653-660 7 Kuster, T., Staudenmann, W., Hughes, G. J. and Heizmann, C. W. (1991) Biochemistry 30, 8812-8816 8 Crumpton, M. J. and Dedman, J. R. (1990) Nature 345, 212 9 Moss, S. E., Edwards, H. E. and Crumpton, M. J. (1991) in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ed.), pp. 535-566, SpringerVerlag 10 Woolgar, J. A., Boustead, C. M. and Walker, J. H. (1990) J. Neurochem. 54, 62-71 11 ReRon, J. K. etal. (1991) J. Exp. Med. 174, 305-310 12 Johnson, M. D., Kamso-Pratt, J. M., Whetsell, W. O. and Pepinsky, R. B. (1989) Am. J. Clin. Pathol. 92,424-429 13 Huber, R. etal. (1992)J. Mol. Biol. 223,683-704 14 Burns, A. L. eta/. (1989) Proc. Natl Acad. Sci. USA 86, 3798-3802 15 Siesj6, B. K. and Wieloch, T. (1986) in Advances in Neurology (Delgado-Escueta, A. V. and Ward, A. A., Jr, eds), pp. 813-847, Raven Press 16 Scharfman, H. E. and Schwartzkroin, P. A. (1989) Science 246, 257-260 17 Meldrum, B. S. (1981) in Metabolic Disorders of the Nervous System (Rose, F. C., ed.), pp. 175-187, Pitman 18 Sloviter, R. S. (1989) J. Neurol. Neurosurg. Psychiatry 45, 1130-1135 19 Leranth, C. and Ribak, C. E. (1991) Exp. Brain Res. 85, 129-136 20 Sloviter, R. S., Solias, A. L., Barbaro, N. M. and Laxer, K. D. (1991) J. Comp. NeuroL 308, 381-396 21 Vonau, M. and T&k, I. (1991) Soc. Neurosci. Abstr. 17, 1260 22 Sloviter, R. S. (1991) Hippocampus 1, 41-66 263
Acknowledgements We apologizeto many colleagues whoseimportant work cou/dnot be dted due to space limitations. We would like to thank A. Rowlersonfor helping with the preparation of this manuscript, M. K#lenfor typing it and K. Wehnerfor photographicalwork. Thisstudy was supportedin part by the SwissNational ScienceFoundation (to C W.H., grant no. 31--30742.91), Hartmann-Miiller Stiftung f6r Medizimsche Forschung, Ciba-
Gegy Jubilaumsstiftung, the Deutsche Bundesministerium f~r Forschungund Technologie,and the Deutsche Forschungsgemeinschaft (to K.B., grant no. Br950/4-5).
23 Kamphuis, W., Huisman, E., Wadman, W. J., Heizmann, C. W. and Lopes da Silva, F. H. (1989) Brain Res. 479, 23-34 24 Lowenstein, D. H., Miles, M. F., Hatam, F. and McCabe, T. (1991) Neuron 6, 627-633 25 Baimbridge, K. G., Mody, I. and Miller, J. J. (1985) Epilepsia 26, 460-465 26 Sonnenberg, J. L. etal. (1991) Mol. Brain Res. 9, 179-190 27 K6hr, G., Lambert, C. E. and Mody, I. (1991) Exp. Brain Res. 85, 543-551 28 Morgan, D. W., Welton, A. F., Heick, A. E. and Christakos, S. (1986) Biochem. Biophys. Res. Commun. 138, 547-553 29 Weiss, J. H., Koh, J. Y., Baimbridge, K. G. and Choi, D. W. (1990) Neurology 40, 1288-1292 30 Waldvogel, H. J., Faull, R. L. M., Williams, M. N. and Dragunow, M. (1991) Brain Res. 546, 329-335 31 Baimbridge, K. G. and Kuo, J. (1988) Soc. Neurosci. Abstr. 14, 1264 32 Winsky, L., Jacobowitz, D. M., Kispert, J. and Henneberry, R. C. (1991) in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ed.), pp. 277-300, Springer-Verlag 33 Johansen, F. F., Tonder, N., Zimmer, J., Baimbridge, K. G. and Diemer, N. H. (1990) Neurosci. Left. 120, 171-174 34 Freund, T. F., Buszaki, G., Leon, A., Baimbridge, K. G. and Somogyi, P. (1990) Exp. Brain Res. 83, 55-66 35 Hof, P. R. et aL (1991)J. NeuropathoL Exp. Neurol. 50, 451-462 36 Iwamoto, N. and Emson, P. C. (1991) Neurosci. Lett. 128, 81-84 37 Arai, H., Emson, P. C., Mountjoy, C. Q., Carassco, L. H. and Heizmann, C. W. (1987) Brain Res. 418, 164-169 38 Satoh, J., Tabira, T., Sano, M,, Nakayama, H. and Tateishi, J. (1991) Acta Neuropathol. 81,388-395 39 McLacHan, D. R. C., Wong, L., Bergeron, C. and Baimbridge, K. G. (1987)Alzheimer Dis. and Assoc. Disord. 1, 171-179 40 Ichimiya, Y., Emson, P. C., Mountjoy, C. Q., Lawson, E. E. M. and Heizmann, C. W. (1988) Brain Res. 475, 156-159 41 lacopino, A. M. and Christakos, S. (1990)J. Biol. Chem. 265, 10177-10180 42 Brady, D. R. and Mufson, E. J. (1991) Soc. Neurosci. Abstr. 17, 691 43 Hof, P. R. and Mordson, J, H. (1991) Exp. Neurol. 111, 293-301 44 Iwamoto, N., Thangnipon, W., Crawford, C. and Emson, P. C. (1991) Brain Res. 561, 177-180 45 Griffin, W. S. eta/. (1989) Proc. Natl Acad. Sci. USA 86, 7611-7615 46 Reeves, R., Crowley, M., Hilt, D. C., Moran, T. and Gerhard, J. (1990) Mouse Molec. Genetics Meeting, Cold Spring Harbor 47 Arai, H., Noguchi, I., Makino, Y., Kosaka, K. and Heizmann,
C. W. (1991) J. Neurol. 238, 200-202 48 Satoh, J., Tabira, T., Sano, M., Nakayama, H. and Tateishi, J. (1991) Acta NeuropathoL 81, 388-395 49 Yamada, T., McGeer, P. L., Baimbridge, K. G. and McGeer, E. G. (1990) Brain Res. 526, 305--307 50 Griffin, W. S. T., Stanley, L. C., Mrak, R. E. and Perrot, L. J. (1991) Soc. Neurosci. Abstr. 17, 1273 51 AIIore, R. et al. (1988)Science 239, 1311-1313 52 Zuckerman, J. E., Herschman, H. R. and Levine, L. (1970) J. Neurochem. 17, 247-251 53 Zimmer, D. B., Cochran, K. H. and Strada, S. J. (1991) Soc. Neurosci. Abstr. 17, 1314 54 Kato, K. eta/. (1990)J. Mol. Neurosci. 2, 109-113 55 Sindic, C. J. M., Chalon, M. P., Cambiaso, C. L., Laterre, E. C. and Masson, P. L. (1982)J. Neurol. Neurosurg. Psychiatry 45, 1130-1135 56 Barger, S. W. and van Eldik, L. J. (1991) Soc. Neurosci. Abstr. 17, 1500 57 Kobayashi, K. et al. (1990) Neurosci. Lett. 113, 17-22 58 Hilt, D. C. and Kligman, D. (1991)in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ecl.), pp. 65-103, Springer-Vedag 59 Persson, L. etal. (1987) Stroke 19, 911-918 60 Ishiguro, Y., Kato, K., Ito, T. and Nagaya, M. (1983) Cancer Res. 43, 6080-6084 61 Fagnart, O. C., Sindic, C. J. M. and Laterre, C. (1988) Clin. Chem. 34, 1387-1391 62 Sofroniew, M. V. and Staley, K. (1991) Trends Neurosci. 14, 513-514 63 Sloviter, R. S. (1987) Science 235, 73-76 64 lacapino, A. M. and Christakos, S. (1990) Proc. Natl Acad. Sci. USA 87, 4078-4082 65 Rogers, J. H. (1991) in Novel Calcium-Binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ed.), pp. 251-276, Springer-Verlag 66 Winsky, L. and Jacobowitz, D. M. (1991) Neurosci. Lett. 131, 79-82 67 Goto, S. eta/. (1988) Exp. Brain Res. 69, 645-650 68 Liu, J. etal. (1991) Cell 66, 807-815 69 Perlmutter, L. S., Gall, C., Baudry, M. and Lynch, G. (1990) J. Comp. Neurol. 296, 269-276 70 Nilson, E., Ostwald, K. and Karlsson, J. O. (1991) Mol. Chem. Neuropathol. 14, 99-105 71 Selinfreund, R. H., Barger, S. W., Pledger, W. J. and van Eldik, L. J. (1991) Proc. NatlAcad. Sci. USA 88, 3554-3558 72 Stryer, L. (1991)J. Biol. Chem. 266, 10711-10714 73 Lambrecht, H-G. and Kock, K-W. (1991) EMBO J. 10, 793-798 74 Polans, A. S., Buczylko, J., Crabb, J. and Palczewski, K (1991) J. Cell. Biol. 112, 981-989 75 Ritzler, J. M. et a/. (1992) Genomics 12, 567-572
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