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Regional specificity of membrane instability in Alzheimer's disease brain
Brain Research, 615 (1993) 355-357
355
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 25715
Regional specificity of membrane instability in Alzheimer's disease brain * Lionel Ginsberg
a J o h n R . A t a c k b,1 S t a n l e y I. R a p o p o r t
b and Norman
L. G e r s h f e l d
a
a Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases and b Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 (USA)
(Accepted 30 March 1993)
Key words: Alzheimer's disease; Membrane; Lipid bilayer instability; Neurodegeneration; Critical temperature
We report an inherent tendency towards the destabilisation of cellular membranes in Alzheimer's disease (AD) brain. This tendency is a natural consequence of abnormal membrane lipid composition, which has previouslybeen documented in AD. Membrane destabilisation may contribute to AD pathogenesis in its own right and may also facilitate amyloid fl-protein deposition, which is potentially neurotoxic. The instability was found to co-localise selectivelywith areas of neurodegeneration in AD brain, thereby possibly accounting for the focal pathology observed in this disorder.
In Alzheimer's disease (AD) there are widespread abnormalities of brain chemistry quite apart from protein anomalies relating to the amyloid plaques and neurofibrillary tangles which are neuropathological hallmarks of the disorder. Thus, for example, prominent changes in m e m b r a n e lipid composition have been observed 1'9'1°. A comprehensive description of A D pathogenesis should establish the relative contributions of all these molecular defects to the neurodegeneration. It should also explain why neuronal death is typically focally distributed in AD, i.e., severely affecting structures such as the temporal lobe but relatively sparing grey matter elsewhere in the brain, e.g., in the cerebellum. We report a spontaneous process of cell m e m b r a n e destabilisation, which is a natural consequence of defective m e m b r a n e lipid composition and which occurs selectively at a site of neurodegeneration in A D brain, thereby potentially accounting for the localised pathology. Lipids in cellular m e m b r a n e s are normally arranged as a bimolecular layer. Rigorous physicochemical studies have established that the assembly and stability of
this bilayer structure are determined by two factors, lipid composition and temperature, which are critically interdependent (reviewed in ref. 2). Thus, lipids extracted from a mammalian neural m e m b r a n e and dispersed in a cell-free system in vitro, will spontaneously form an isolated bilayer state, resembling their structure in the original membrane. But this will only occur at a single critical temperature, T * , which normally equals the physiological temperature, Tp ( = 37°C), of the source m e m b r a n e in vivo 4. At other temperatures, the equilibrium lipid state is generally multilamellar 2. A homeostatic mechanism is therefore envisaged in normal cells which maintains a critical lipid composition appropriate for spontaneous bilayer assembly at Tf'4. D e p a r t u r e from this critical composition, such that T * no longer equals Tp, can produce m e m b r a n e destabilisation, hence cellular damage and ultimately death 2'4. We have previously shown that a primary metabolic defect which alters m e m b r a n e lipid composition in a neurological disorder, can shift T * significantly from 37°C. M e m b r a n e destabilisation would then be predicted, providing a potential link between the
Correspondence: L. Ginsberg. Present address: Department of Neuroscience, Royal Free Hospital School of Medicine, Rowland Hill Street,
London NW3 2PF, UK. Fax: (44) (71) 431-1577. 1 Present address: Merck Sharp and Dohme Neuroscience Research Centre, Harlow, (UK). * Presented in part at a symposiumon Molecular Science in Neurodegeneration and Regeneration, Bristol, September 1991.
356 TABLE I
initial biochemical abnormality and the pathological consequences 3. We report here measurements of T* for membrane lipids from different regions of AD brain. Brain tissue was obtained at necropsy from three patients who had died with AD, diagnosed clinically and confirmed neuropathologically (aged 73-83 years, one female, delay from death to brain removal < 9 h), and from four matched control individuals (aged 73-78 years, one female, post-mortem delay < 14 h). Controls had no clinical or pathological evidence of neurological disease. Their nutritional and metabolic status immediately ante mortem did, however, in some cases correspond to that anticipated in AD (a 'catabolic' state reflecting intercurrent infection and malnutrition). For example, one control patient (whose results are shown in Fig. 1) had died from the consequences of stage IVB Hodgkin's disease. The value of Tp was 37°C in all cases, i.e., there was no evidence of hypothermia even terminally in any of the patients' hospital records. Coronal slices of brain tissue were frozen and stored at - 80°C before dissection and lipid extraction. The regions studied were grey matter from cerebral cortex (mid-temporal lobe) and cerebellum. Total lipid extracts from these regions were prepared using organic solvents, including in some instances rigorous purification to remove protein traces by a solid phase extraction technique 4. The method of determining T* for these lipid extracts and illustrative results, are shown in Fig. 1.
Values of the critical temperature, T *, Jbr total lipid extracts from grey matter of Alzheimer's disease (AD) patients and control individuals Subject (sex / age, years)
Neuropathological diagnosis
F/75 M/76
Control Control
M/78 M/73 M/73
Control Control AD
F/77 M/83
AD AD
T* cerebral cortex (mid-temporal)
cerebellar grey matter
(°c)
(oc)
36 _+ 1 35 +_ 1 38+ I # 36 ± 1 # 19_+. 1 20_+3 # 26 ± 2 28 ± 1
37 ± 1 37 ± 1 37+ 1 # 36 ± 1 # 37+ 1 37_+1 # 37 + 1 38 ± 2
# Lipid extracts rigorously purified to remove protein traces. 4
Membrane lipids from mid-temporal cortex of AD brains, a site of neurodegeneration, consistently gave values of T* significantly below Tp ( T * = 19-28°C). But lipids from cerebellar grey matter, relatively spared in AD, gave a normal value for T*, i.e., approximating Tp = 37°C, as did grey matter lipids from the controls, whether from mid-temporal cortex or cerebellum (Fig. 1, Table I). These normal results for the control brains, with a similar delay between death and brain removal to the AD samples and for the internal control of cerebellum from AD patients, eliminate the possibility that the findings in AD mid-temporal cortex were merely a spurious effect of post mortem tissue autoly-
Female, 75yrs Normal
Male, 73yrs Alzhelmer's 37° ~.
40-
E E
z
35-
E
35_
30--
30-
25-
I
10
I
I
I
20 30 40 Temperature (°C)
I
10
I
i
I
20 30 40 Temperature (°C)
Fig. 1. Measurement of the critical temperature, T*, for total lipid extracts from human brain grey matter in health and disease. In a cell-free system, the temperature, T*, at which an equilibrium aqueous dispersion of a brain lipid extract forms the 'critical bilayer state', equivalent to the structure in cell membranes, equals the temperature at which the surface pressure ( % ) of the dispersion is maximal. The theoretical and experimental rationale for this surface approach, which is empirically more accessible than bulk lipid measurements, is discussed in detail elsewhere 2'4. Each data point in these surface pressure-temperature phase diagrams was obtained independently with a fresh lipid preparation. ,x = mid-temporal cortex; • = cerebellar grey matter.
357 sis. The T * results for AD mid-temporal cortex were also unlikely to be secondary to age or metabolic state as the controls were matched for these variables. Furthermore, our previous studies indicate that a drop in T * would not occur simply secondary to extensive neurodegeneration in an affected brain region. A change in total lipid composition in such a region due to neuronal damage and replacement by other cell types would not necessarily be expected to change T * on theoretical grounds 2. Furthermore, in the case of lipids from plaques of multiple sclerosis where total lipid composition is indeed abnormal due to myelin loss and extensive gliosis, we have previously shown that T * remains normal at 37°C 3. The finding that T * < Tp in AD mid-temporal cortex but not cerebellar grey matter indicates a tendency towards membrane bilayer destabilisation and therefore cellular injury selectively at a tissue site affected by the disease. This shift in T * for AD mid-temporal cortex implies a lipid compositional abnormality within cell membranes at that site. Membrane lipid anomalies have previously been reported in AD 1'9'1°. The critical bilayer mechanism gives a logical basis to the proposed pathogenetic effect of such anomalies and can account for the disease localisation. The cause of a localised membrane lipid compositional change is uncertain, though the relevant defect may relate to a biochemical pathway of greater significance in cerebral cortical rather than cerebellar neurons. Alternatively, different regions of the brain may possess differing abilities to compensate for a lipid metabolic defect 3. Membrane destabilisation may contribute to the development of AD independently of other proposed pathogenetic pathways, e.g., amyloid /3-protein deposition 5. But these two apparently disparate processes may act synergistically. Amyloid/3-protein is formed by enzymatic cleavage of a membrane-spanning precursor protein (APP) within its transmembrane portion and it
is currently unclear how this occurs 7. We suggest that amyloidogenesis may occur when APP is rendered susceptible to aberrant enzymatic proteolysis by itself being abnormal, either qualitatively, due to a mutation in the APP gene 6 or quantitatively, as may be the case in Down's syndrome due to a gene dosage effect. Alternatively, normal APP may become unusually accessible to amyloidogenic proteolytic attack by destabilisation of the surrounding membrane as postulated elsewhere 8'9 and indicated by our results. We thank W.F. Stevens Jr. for technical assistance and Dr. Daniel Brady for confirming the neuropathological diagnoses. 1 Ellison, D.W., Beal, M.F. and Martin, J.B., Phosphoethanolamine and ethanolamine are decreased in Alzheimer's disease and Huntington's disease, Brain Res., 417 (1987) 389-392. 2 Gershfeld, N.L., The critical unilamellar lipid state: a perspective for membrane bilayer assembly, Bioehim. Biophys. Acta, 988 (1989) 335-350. 3 Ginsberg, L. and Gershfeld, N.L., Membrane bilayer instability and the pathogenesis of disorders of myelin. Neurosei. Lett., 130 (1991) 133-136. 4 Ginsberg, L., Gilbert, D.L. and Gershfeld, N.L., Membrane bilayer assembly in neural tissue of rat and squid as a critical phenomenon: influence of temperature and membrane proteins, J. Membr. Biol., 119 (1991) 65-73. 5 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 6 Goate, A., Chartier-Harlin, M.-C., Mullan, M., Brown, J., Crawford, F., Fidani, L., et al., Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease, Nature, 349 (1991) 704-706. 7 Hardy, J. and Mullan, M., Alzheimer's disease: in search of the soluble, Nature, 359 (1992) 268-269. 8 Miiller-Hill, B. and Beyreuther, K., Molecular biology of Alzheimer's disease, Annu. Rev. Biochem., 58 (1989) 287-307. 9 Nitsch, R.M., Blusztajn, J.K., Pittas, A.G., Slack, B.E., Growdon, J.H. and Wurtman, R.J., Evidence for a membrane defect in Alzheimer disease brain, Proc. Natl. Acad. Sci. USA, 89 (1992) 1671-1675. 10 Pettegrew, J.W., Moossy, J., Withers, G., McKeag, D. and Panchalingam, K., 31p nuclear magnetic resonance study of the brain in Alzheimer's disease, J. Neuropathol. Exp. Neurol. 47 (1988) 235-48.
355
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 25715
Regional specificity of membrane instability in Alzheimer's disease brain * Lionel Ginsberg
a J o h n R . A t a c k b,1 S t a n l e y I. R a p o p o r t
b and Norman
L. G e r s h f e l d
a
a Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases and b Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 (USA)
(Accepted 30 March 1993)
Key words: Alzheimer's disease; Membrane; Lipid bilayer instability; Neurodegeneration; Critical temperature
We report an inherent tendency towards the destabilisation of cellular membranes in Alzheimer's disease (AD) brain. This tendency is a natural consequence of abnormal membrane lipid composition, which has previouslybeen documented in AD. Membrane destabilisation may contribute to AD pathogenesis in its own right and may also facilitate amyloid fl-protein deposition, which is potentially neurotoxic. The instability was found to co-localise selectivelywith areas of neurodegeneration in AD brain, thereby possibly accounting for the focal pathology observed in this disorder.
In Alzheimer's disease (AD) there are widespread abnormalities of brain chemistry quite apart from protein anomalies relating to the amyloid plaques and neurofibrillary tangles which are neuropathological hallmarks of the disorder. Thus, for example, prominent changes in m e m b r a n e lipid composition have been observed 1'9'1°. A comprehensive description of A D pathogenesis should establish the relative contributions of all these molecular defects to the neurodegeneration. It should also explain why neuronal death is typically focally distributed in AD, i.e., severely affecting structures such as the temporal lobe but relatively sparing grey matter elsewhere in the brain, e.g., in the cerebellum. We report a spontaneous process of cell m e m b r a n e destabilisation, which is a natural consequence of defective m e m b r a n e lipid composition and which occurs selectively at a site of neurodegeneration in A D brain, thereby potentially accounting for the localised pathology. Lipids in cellular m e m b r a n e s are normally arranged as a bimolecular layer. Rigorous physicochemical studies have established that the assembly and stability of
this bilayer structure are determined by two factors, lipid composition and temperature, which are critically interdependent (reviewed in ref. 2). Thus, lipids extracted from a mammalian neural m e m b r a n e and dispersed in a cell-free system in vitro, will spontaneously form an isolated bilayer state, resembling their structure in the original membrane. But this will only occur at a single critical temperature, T * , which normally equals the physiological temperature, Tp ( = 37°C), of the source m e m b r a n e in vivo 4. At other temperatures, the equilibrium lipid state is generally multilamellar 2. A homeostatic mechanism is therefore envisaged in normal cells which maintains a critical lipid composition appropriate for spontaneous bilayer assembly at Tf'4. D e p a r t u r e from this critical composition, such that T * no longer equals Tp, can produce m e m b r a n e destabilisation, hence cellular damage and ultimately death 2'4. We have previously shown that a primary metabolic defect which alters m e m b r a n e lipid composition in a neurological disorder, can shift T * significantly from 37°C. M e m b r a n e destabilisation would then be predicted, providing a potential link between the
Correspondence: L. Ginsberg. Present address: Department of Neuroscience, Royal Free Hospital School of Medicine, Rowland Hill Street,
London NW3 2PF, UK. Fax: (44) (71) 431-1577. 1 Present address: Merck Sharp and Dohme Neuroscience Research Centre, Harlow, (UK). * Presented in part at a symposiumon Molecular Science in Neurodegeneration and Regeneration, Bristol, September 1991.
356 TABLE I
initial biochemical abnormality and the pathological consequences 3. We report here measurements of T* for membrane lipids from different regions of AD brain. Brain tissue was obtained at necropsy from three patients who had died with AD, diagnosed clinically and confirmed neuropathologically (aged 73-83 years, one female, delay from death to brain removal < 9 h), and from four matched control individuals (aged 73-78 years, one female, post-mortem delay < 14 h). Controls had no clinical or pathological evidence of neurological disease. Their nutritional and metabolic status immediately ante mortem did, however, in some cases correspond to that anticipated in AD (a 'catabolic' state reflecting intercurrent infection and malnutrition). For example, one control patient (whose results are shown in Fig. 1) had died from the consequences of stage IVB Hodgkin's disease. The value of Tp was 37°C in all cases, i.e., there was no evidence of hypothermia even terminally in any of the patients' hospital records. Coronal slices of brain tissue were frozen and stored at - 80°C before dissection and lipid extraction. The regions studied were grey matter from cerebral cortex (mid-temporal lobe) and cerebellum. Total lipid extracts from these regions were prepared using organic solvents, including in some instances rigorous purification to remove protein traces by a solid phase extraction technique 4. The method of determining T* for these lipid extracts and illustrative results, are shown in Fig. 1.
Values of the critical temperature, T *, Jbr total lipid extracts from grey matter of Alzheimer's disease (AD) patients and control individuals Subject (sex / age, years)
Neuropathological diagnosis
F/75 M/76
Control Control
M/78 M/73 M/73
Control Control AD
F/77 M/83
AD AD
T* cerebral cortex (mid-temporal)
cerebellar grey matter
(°c)
(oc)
36 _+ 1 35 +_ 1 38+ I # 36 ± 1 # 19_+. 1 20_+3 # 26 ± 2 28 ± 1
37 ± 1 37 ± 1 37+ 1 # 36 ± 1 # 37+ 1 37_+1 # 37 + 1 38 ± 2
# Lipid extracts rigorously purified to remove protein traces. 4
Membrane lipids from mid-temporal cortex of AD brains, a site of neurodegeneration, consistently gave values of T* significantly below Tp ( T * = 19-28°C). But lipids from cerebellar grey matter, relatively spared in AD, gave a normal value for T*, i.e., approximating Tp = 37°C, as did grey matter lipids from the controls, whether from mid-temporal cortex or cerebellum (Fig. 1, Table I). These normal results for the control brains, with a similar delay between death and brain removal to the AD samples and for the internal control of cerebellum from AD patients, eliminate the possibility that the findings in AD mid-temporal cortex were merely a spurious effect of post mortem tissue autoly-
Female, 75yrs Normal
Male, 73yrs Alzhelmer's 37° ~.
40-
E E
z
35-
E
35_
30--
30-
25-
I
10
I
I
I
20 30 40 Temperature (°C)
I
10
I
i
I
20 30 40 Temperature (°C)
Fig. 1. Measurement of the critical temperature, T*, for total lipid extracts from human brain grey matter in health and disease. In a cell-free system, the temperature, T*, at which an equilibrium aqueous dispersion of a brain lipid extract forms the 'critical bilayer state', equivalent to the structure in cell membranes, equals the temperature at which the surface pressure ( % ) of the dispersion is maximal. The theoretical and experimental rationale for this surface approach, which is empirically more accessible than bulk lipid measurements, is discussed in detail elsewhere 2'4. Each data point in these surface pressure-temperature phase diagrams was obtained independently with a fresh lipid preparation. ,x = mid-temporal cortex; • = cerebellar grey matter.
357 sis. The T * results for AD mid-temporal cortex were also unlikely to be secondary to age or metabolic state as the controls were matched for these variables. Furthermore, our previous studies indicate that a drop in T * would not occur simply secondary to extensive neurodegeneration in an affected brain region. A change in total lipid composition in such a region due to neuronal damage and replacement by other cell types would not necessarily be expected to change T * on theoretical grounds 2. Furthermore, in the case of lipids from plaques of multiple sclerosis where total lipid composition is indeed abnormal due to myelin loss and extensive gliosis, we have previously shown that T * remains normal at 37°C 3. The finding that T * < Tp in AD mid-temporal cortex but not cerebellar grey matter indicates a tendency towards membrane bilayer destabilisation and therefore cellular injury selectively at a tissue site affected by the disease. This shift in T * for AD mid-temporal cortex implies a lipid compositional abnormality within cell membranes at that site. Membrane lipid anomalies have previously been reported in AD 1'9'1°. The critical bilayer mechanism gives a logical basis to the proposed pathogenetic effect of such anomalies and can account for the disease localisation. The cause of a localised membrane lipid compositional change is uncertain, though the relevant defect may relate to a biochemical pathway of greater significance in cerebral cortical rather than cerebellar neurons. Alternatively, different regions of the brain may possess differing abilities to compensate for a lipid metabolic defect 3. Membrane destabilisation may contribute to the development of AD independently of other proposed pathogenetic pathways, e.g., amyloid /3-protein deposition 5. But these two apparently disparate processes may act synergistically. Amyloid/3-protein is formed by enzymatic cleavage of a membrane-spanning precursor protein (APP) within its transmembrane portion and it
is currently unclear how this occurs 7. We suggest that amyloidogenesis may occur when APP is rendered susceptible to aberrant enzymatic proteolysis by itself being abnormal, either qualitatively, due to a mutation in the APP gene 6 or quantitatively, as may be the case in Down's syndrome due to a gene dosage effect. Alternatively, normal APP may become unusually accessible to amyloidogenic proteolytic attack by destabilisation of the surrounding membrane as postulated elsewhere 8'9 and indicated by our results. We thank W.F. Stevens Jr. for technical assistance and Dr. Daniel Brady for confirming the neuropathological diagnoses. 1 Ellison, D.W., Beal, M.F. and Martin, J.B., Phosphoethanolamine and ethanolamine are decreased in Alzheimer's disease and Huntington's disease, Brain Res., 417 (1987) 389-392. 2 Gershfeld, N.L., The critical unilamellar lipid state: a perspective for membrane bilayer assembly, Bioehim. Biophys. Acta, 988 (1989) 335-350. 3 Ginsberg, L. and Gershfeld, N.L., Membrane bilayer instability and the pathogenesis of disorders of myelin. Neurosei. Lett., 130 (1991) 133-136. 4 Ginsberg, L., Gilbert, D.L. and Gershfeld, N.L., Membrane bilayer assembly in neural tissue of rat and squid as a critical phenomenon: influence of temperature and membrane proteins, J. Membr. Biol., 119 (1991) 65-73. 5 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 6 Goate, A., Chartier-Harlin, M.-C., Mullan, M., Brown, J., Crawford, F., Fidani, L., et al., Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease, Nature, 349 (1991) 704-706. 7 Hardy, J. and Mullan, M., Alzheimer's disease: in search of the soluble, Nature, 359 (1992) 268-269. 8 Miiller-Hill, B. and Beyreuther, K., Molecular biology of Alzheimer's disease, Annu. Rev. Biochem., 58 (1989) 287-307. 9 Nitsch, R.M., Blusztajn, J.K., Pittas, A.G., Slack, B.E., Growdon, J.H. and Wurtman, R.J., Evidence for a membrane defect in Alzheimer disease brain, Proc. Natl. Acad. Sci. USA, 89 (1992) 1671-1675. 10 Pettegrew, J.W., Moossy, J., Withers, G., McKeag, D. and Panchalingam, K., 31p nuclear magnetic resonance study of the brain in Alzheimer's disease, J. Neuropathol. Exp. Neurol. 47 (1988) 235-48.