~~ ; ~
~
~
~
~
~
~
;
~
meeting
report
t is hard to imagine a more Iin.spectacular venue than Big Sky, Alzheimer's diseaseand flA4: getting to the the heart of the Montana mountains, for the recent Keystone symposium on neurodegenerative disease*. The week-long meeting, organized by Drs Cunningham and Price, attracted top researchers from all over the USA as well as a small contingent from Europe. The relaxed atmosphere and superb location were conducive to extensive and sometimes heated scientific debate. The proceedings commenced with a keynote address by Dr Carlton Gajdusek, who provided an overview of amyloidogenesis in Alzheimer's disease (AD) and the spongiform encephalopathies. Perhaps not surprisingly, considering the current interest in AD, it was discussion of this disorder that dominated the meeting. In particular, the roles of ~ amyloid ([3A4) and its precursor protein ([3APP) were the focus of much attention. The main protein constituent of the plaques and cerebrovascular lesions found in the brains of patients with AD is ~A4. This protein is formed as a result of abnormal processing of its precursor protein, [3APP. Normal processing of this protein, which takes place on the plasma membrane, involves a specific cleavage of the peptide chain and the subsequent production of a secreted fragment. An elegant set of experiments demonstrated that the action of the enzyme involved in [~APP secretion is not dependent on the amino acid sequence of the target protein, but instead depends on the a-helical structure of the protein near the membrane, and on the distance of the cleavage site from the membrane (Sangram Sisodia, Johns Hopkins University School of Medicine, USA). Normal cleavage takes place at a site within the ~A4 protein sequence, which effectively dismisses this process as a means of producing intact [3A4. Evidence was put forward for an alternative pathway of [3APP metabolism (Dennis Selkoe, Harvard University Medical School, USA; Steven Younkin, Case Western Reserve Univer-
core of the problem?
sity, USA) that would allow the generation of peptide fragments containing the full [~A4 sequence. The pathway involves the internalization of the precursor protein into the lysosomal system of the cell, which can be demonstrated by labelling cell-surface [3APP with a specific antibody and observing the internalization of antibody-antigen complexes (Selkoe). This process results in a complex mix of carboxy-terminal fragments in the cell, some of which contain the potentially amyloidogenic [3A4 peptide (Younkin). This idea was strengthened fln-ther by the characterization of a sequence of six amino acids within the carboxyterminal of the ~APP molecule that is essential for rapid endocytosis of the protein (Sisodia). The normal cellular role of [3APP remains unresolved, but, based on work done on the expression of the protein in monocytes, it was suggested that this protein may be involved in cell adhesion (Konrad Beyreuther, University of Heidelberg, FRG). It was hypothesized that the formation of [3A4 in the brain may interfere with the binding of [3APP to the extracellular matrix in synapses. Studies carried out in cell culture produced a somewhat confused picture of the mechanism of [3A4 toxicity. This is probably due to different labs using different culture systems. The toxic effects of [3A4 in some systems do not appear to be mediated by glutamate (Bruce Yankner, Children's Hospital, Boston, USA), while in others, the peptide enhances the vulnerability of neurons to excitotoxic damage (Carl Cotman, University of California, Irvine, USA). It is interesting to note that the toxicity of the peptide is affected by its age and aggregation state (Cotman). A remarkable series of timecourse experiments using a combination of methodologies, namely cell culture and doublelabelling immunocytochemistry, provided some compelling evidence *Advances in Understanding Neuro- that [3A4 plays a role in the inducdegenerative Disorders. Held in Big Sky, tion of neurofibrillary pathology (Yankner). MT, USA: 28 March- 4 April 1992.
TINS, Vol. 15, No. 9, 1992
© 1992, Elsevier Science Publishers Ltd, (UK)
A further contentious issue concerning [3A4 is the question of its cellular origin. One suggestion, based on extensive electron microscope studies on biopsy material, is that [3A4-containing amyloid fibrils are formed by the processing of [3APP in microglia (Henry Wisniewski, New York State Institute for Basic Research in Developmental Disabilities, USA). On the other hand, studies in primates have shown that [3APP is axonally transported in neurons and processed in the terminal fields (Donald Price, Johns Hopkins University School of Medicine, USA). This would account for the synaptic pathology associated with AD. While these two options are not mutually exclusive, as both cell types could produce [~A4, the balance of opinion currently favours the latter possibility. The secreted fragment of [3APP, which contains the Kunitz protease inhibitor domain, is homologous to protease nexin 2 (PN-2). Levels of this protein were reported to be reduced in the cerebrospinal fluid (CSF) of patients with suspected AD and in patients with Dutch hereditary amyloidosis (HCHWA-D) (William Van Nostrand, University of California, Irvine, USA). Of particular interest in this study was the fact that patients with familial AD who had both an identified point mutation in their [3APP gene sequence and severe congophilic angiopathy showed a greater reduction in CSF levels of the secreted protein than patients who had the mutation alone. It therefore appears that a pronounced reduction of [3APP in the CSF is a common feature of disorders that involve cerebrovascular deposition of [3A4. Results of studies with the potent thrombin inhibitor protease nexin 1 (PN-1) were also presented (Dennis Cunningham, University of California, Irvine, USA). PN-1 is located in astrocytes, capillaries and larger blood vessels, and its activity is markedly reduced in AD. This vascular localization indicates that the protein may protect against changes
Stephen M. Gentleman Deptof Anatomy, St Mary'sMedical School,Imperial College,Norfolk Place,London,UK W2 1PG
31 5
in the blood-brain barrier. It was proposed that a reduction in the level of PN-1 causes disruption of the blood-brain barrier, increased thrombin levels, and ultimately damage to nearby neurons and astrocytes. The reason for the observed reduction in the level of PN-1 in AD remains unclear. Other aspects of AD that were addressed during the meeting included a session on the neurofibfillary abnormalities associated with the disease, and another on the growing interest in the possible role of neurotrophic factors in the treatment of AD. In the former session, as with the discussions on ~A4 and plaques, attention focused very much on a single protein, in this case the microtubule-associated protein tau, and its role in the formation of neurofibrillary tangles. Abnormal phosphorylafion of tau is thought to lead to the formation of the paired helical filaments that are observed in tangles (Virginia Lee, University of Pennsylvania School of Medicine, USA; Khalid Iqbal, New York State Institute for Basic Research in Developmental Disabilities, USA), although phosphorylafion alone may not be enough to cause this pathology (Kenneth Kosik, Harvard University, USA). It was also suggested that the presence of abnormal tau may be more closely correlated with the presence of dystrophic neurites than with tangles (Claude Wischik, University of Cambridge, UK). Neurotrophic factors regulate the growth, plasticity and regeneration of neurons. These properties have made them particularly attractive as potential therapeutic agents to combat degenerative disease. The most potent of these is nerve growth factor (NGF), which promotes neuronal survival and neurite outgrowth. However, its lack of specificity for defined cell populations, and problems with functional hyperactivity and aberrant neuron growth all make NGF less than ideal for use as a treatment in AD (Franz Hefti, University of Southern California, USA). Therefore the search is currently underway for more specific neurotrophic factors. However, this has produced mixed results. Brain-derived neurotrophic factor has protective actions in the forebrain, but is not as effective as NGF (Hefti), while 316
ciliary neurotrophic factor has a their attempts at producing transprofound rescue effect on degen- genic animals using a variety of erating thalamic neurons (Vassilis genetic inserts, some of which Koliatsos, Johns Hopkins Univer- contained the identified mutations. sity School of Medicine, USA). The overwhelming impression Although much of the meeting given was that although this is a concentrated on AD, there was a valuable avenue of research, which sprinkling of presentations on offers by far the best hope for a other disorders, most notably viable animal model of AD, there the spongiform encephalopathies. are still technical difficulties and Clinical and molecular genetic problems to be overcome. Not studies of Creutzfeldt-Jakob dis- least of these appears to be that ease show that all familial cases of only very low levels of the proteins the disease have a mutation in the encoded by the transgenes are prion protein gene, most com- obtained, even when expression of monly in codon 200 (Lev Goldfarb, the transgenes themselves is high. National Institutes of Health, The overall impression left by USA). Progress in the production the meeting was one of cautious of a transgenic model of the dis- optimism - that perhaps there is ease was also discussed (Karen light at the end of the tunnel. Over Hsiao, University of California, San the past few years there seems to Francisco, USA). Intriguing ques- have been a convergence of ideas tions remain as to how different and of apparently disparate restrains of scrapie, the sheep equiv- search strategies. Whether invesalent of Creutzfeldt-Jakob disease, tigations have been based on produce strain-specific pathologies genetics, cell biology or, as in my and target different areas of the own case, a link with head injury, a brain (Steve de Armond, Univer- large number of groups have come sity of California, San Francisco, to think of [3APP as being central USA). to unravelling the mechanisms of The meeting ended with an im- AD. This meeting provided a valupromptu session on the current able insight into what is now known state of transgenic animal re- about the processing and function search. The session opened with of ~IAPP and, as with all good Alison Goate (St Mary's Medical meetings, it raised just as many School, London, UK) giving a brief questions as it answered. I for one summary of the 717 mutations came away with a wealth of new identified in the [3APP gene. A ideas and a renewed enthusiasm to string of speakers then described get back into the laboratory.
Trends in Neurosciences
Subscriptions
All new subscriptions, renewals and subscription queries should be addressed to our Barking office at the address below.
[] Personal subscriptions (1992, 12 monthly issues): £59.00 in the UK; $92.00 in the USA; £63.00 elsewhere [] Library subscriptions (1992, 12 monthly issues plus hard-cover bound compendium with index): £239.00 in the UK and ROW [] Student subscriptions (1992, 12 monthly issues): £49.00 in the U K; $77.00 in the USA; £53.00 elsewhere (Student subscriptions must be accompanied by valid proof of student status.) All prices include postage (air delivery outside UK). All subscriptions can be paid for by cheque, bank draft or UNESCO coupons. Personal and Library subscriptions can also be paid for by credit card - Access/ Mastercard, Eurocard, Visa or American Express. Subscription orders should be sent to: Elsevier Science Publishers Ltd, Crown House, Linton Road, Barking, UK I G l l 8JU. Tel. (outside UK): +44 81 5947272. TeL On UK): 081 594 7272
TINS, VoL 15, No. 9, 1992
~
~
~
~
~
~
;
~
meeting
report
t is hard to imagine a more Iin.spectacular venue than Big Sky, Alzheimer's diseaseand flA4: getting to the the heart of the Montana mountains, for the recent Keystone symposium on neurodegenerative disease*. The week-long meeting, organized by Drs Cunningham and Price, attracted top researchers from all over the USA as well as a small contingent from Europe. The relaxed atmosphere and superb location were conducive to extensive and sometimes heated scientific debate. The proceedings commenced with a keynote address by Dr Carlton Gajdusek, who provided an overview of amyloidogenesis in Alzheimer's disease (AD) and the spongiform encephalopathies. Perhaps not surprisingly, considering the current interest in AD, it was discussion of this disorder that dominated the meeting. In particular, the roles of ~ amyloid ([3A4) and its precursor protein ([3APP) were the focus of much attention. The main protein constituent of the plaques and cerebrovascular lesions found in the brains of patients with AD is ~A4. This protein is formed as a result of abnormal processing of its precursor protein, [3APP. Normal processing of this protein, which takes place on the plasma membrane, involves a specific cleavage of the peptide chain and the subsequent production of a secreted fragment. An elegant set of experiments demonstrated that the action of the enzyme involved in [~APP secretion is not dependent on the amino acid sequence of the target protein, but instead depends on the a-helical structure of the protein near the membrane, and on the distance of the cleavage site from the membrane (Sangram Sisodia, Johns Hopkins University School of Medicine, USA). Normal cleavage takes place at a site within the ~A4 protein sequence, which effectively dismisses this process as a means of producing intact [3A4. Evidence was put forward for an alternative pathway of [3APP metabolism (Dennis Selkoe, Harvard University Medical School, USA; Steven Younkin, Case Western Reserve Univer-
core of the problem?
sity, USA) that would allow the generation of peptide fragments containing the full [~A4 sequence. The pathway involves the internalization of the precursor protein into the lysosomal system of the cell, which can be demonstrated by labelling cell-surface [3APP with a specific antibody and observing the internalization of antibody-antigen complexes (Selkoe). This process results in a complex mix of carboxy-terminal fragments in the cell, some of which contain the potentially amyloidogenic [3A4 peptide (Younkin). This idea was strengthened fln-ther by the characterization of a sequence of six amino acids within the carboxyterminal of the ~APP molecule that is essential for rapid endocytosis of the protein (Sisodia). The normal cellular role of [3APP remains unresolved, but, based on work done on the expression of the protein in monocytes, it was suggested that this protein may be involved in cell adhesion (Konrad Beyreuther, University of Heidelberg, FRG). It was hypothesized that the formation of [3A4 in the brain may interfere with the binding of [3APP to the extracellular matrix in synapses. Studies carried out in cell culture produced a somewhat confused picture of the mechanism of [3A4 toxicity. This is probably due to different labs using different culture systems. The toxic effects of [3A4 in some systems do not appear to be mediated by glutamate (Bruce Yankner, Children's Hospital, Boston, USA), while in others, the peptide enhances the vulnerability of neurons to excitotoxic damage (Carl Cotman, University of California, Irvine, USA). It is interesting to note that the toxicity of the peptide is affected by its age and aggregation state (Cotman). A remarkable series of timecourse experiments using a combination of methodologies, namely cell culture and doublelabelling immunocytochemistry, provided some compelling evidence *Advances in Understanding Neuro- that [3A4 plays a role in the inducdegenerative Disorders. Held in Big Sky, tion of neurofibrillary pathology (Yankner). MT, USA: 28 March- 4 April 1992.
TINS, Vol. 15, No. 9, 1992
© 1992, Elsevier Science Publishers Ltd, (UK)
A further contentious issue concerning [3A4 is the question of its cellular origin. One suggestion, based on extensive electron microscope studies on biopsy material, is that [3A4-containing amyloid fibrils are formed by the processing of [3APP in microglia (Henry Wisniewski, New York State Institute for Basic Research in Developmental Disabilities, USA). On the other hand, studies in primates have shown that [3APP is axonally transported in neurons and processed in the terminal fields (Donald Price, Johns Hopkins University School of Medicine, USA). This would account for the synaptic pathology associated with AD. While these two options are not mutually exclusive, as both cell types could produce [~A4, the balance of opinion currently favours the latter possibility. The secreted fragment of [3APP, which contains the Kunitz protease inhibitor domain, is homologous to protease nexin 2 (PN-2). Levels of this protein were reported to be reduced in the cerebrospinal fluid (CSF) of patients with suspected AD and in patients with Dutch hereditary amyloidosis (HCHWA-D) (William Van Nostrand, University of California, Irvine, USA). Of particular interest in this study was the fact that patients with familial AD who had both an identified point mutation in their [3APP gene sequence and severe congophilic angiopathy showed a greater reduction in CSF levels of the secreted protein than patients who had the mutation alone. It therefore appears that a pronounced reduction of [3APP in the CSF is a common feature of disorders that involve cerebrovascular deposition of [3A4. Results of studies with the potent thrombin inhibitor protease nexin 1 (PN-1) were also presented (Dennis Cunningham, University of California, Irvine, USA). PN-1 is located in astrocytes, capillaries and larger blood vessels, and its activity is markedly reduced in AD. This vascular localization indicates that the protein may protect against changes
Stephen M. Gentleman Deptof Anatomy, St Mary'sMedical School,Imperial College,Norfolk Place,London,UK W2 1PG
31 5
in the blood-brain barrier. It was proposed that a reduction in the level of PN-1 causes disruption of the blood-brain barrier, increased thrombin levels, and ultimately damage to nearby neurons and astrocytes. The reason for the observed reduction in the level of PN-1 in AD remains unclear. Other aspects of AD that were addressed during the meeting included a session on the neurofibfillary abnormalities associated with the disease, and another on the growing interest in the possible role of neurotrophic factors in the treatment of AD. In the former session, as with the discussions on ~A4 and plaques, attention focused very much on a single protein, in this case the microtubule-associated protein tau, and its role in the formation of neurofibrillary tangles. Abnormal phosphorylafion of tau is thought to lead to the formation of the paired helical filaments that are observed in tangles (Virginia Lee, University of Pennsylvania School of Medicine, USA; Khalid Iqbal, New York State Institute for Basic Research in Developmental Disabilities, USA), although phosphorylafion alone may not be enough to cause this pathology (Kenneth Kosik, Harvard University, USA). It was also suggested that the presence of abnormal tau may be more closely correlated with the presence of dystrophic neurites than with tangles (Claude Wischik, University of Cambridge, UK). Neurotrophic factors regulate the growth, plasticity and regeneration of neurons. These properties have made them particularly attractive as potential therapeutic agents to combat degenerative disease. The most potent of these is nerve growth factor (NGF), which promotes neuronal survival and neurite outgrowth. However, its lack of specificity for defined cell populations, and problems with functional hyperactivity and aberrant neuron growth all make NGF less than ideal for use as a treatment in AD (Franz Hefti, University of Southern California, USA). Therefore the search is currently underway for more specific neurotrophic factors. However, this has produced mixed results. Brain-derived neurotrophic factor has protective actions in the forebrain, but is not as effective as NGF (Hefti), while 316
ciliary neurotrophic factor has a their attempts at producing transprofound rescue effect on degen- genic animals using a variety of erating thalamic neurons (Vassilis genetic inserts, some of which Koliatsos, Johns Hopkins Univer- contained the identified mutations. sity School of Medicine, USA). The overwhelming impression Although much of the meeting given was that although this is a concentrated on AD, there was a valuable avenue of research, which sprinkling of presentations on offers by far the best hope for a other disorders, most notably viable animal model of AD, there the spongiform encephalopathies. are still technical difficulties and Clinical and molecular genetic problems to be overcome. Not studies of Creutzfeldt-Jakob dis- least of these appears to be that ease show that all familial cases of only very low levels of the proteins the disease have a mutation in the encoded by the transgenes are prion protein gene, most com- obtained, even when expression of monly in codon 200 (Lev Goldfarb, the transgenes themselves is high. National Institutes of Health, The overall impression left by USA). Progress in the production the meeting was one of cautious of a transgenic model of the dis- optimism - that perhaps there is ease was also discussed (Karen light at the end of the tunnel. Over Hsiao, University of California, San the past few years there seems to Francisco, USA). Intriguing ques- have been a convergence of ideas tions remain as to how different and of apparently disparate restrains of scrapie, the sheep equiv- search strategies. Whether invesalent of Creutzfeldt-Jakob disease, tigations have been based on produce strain-specific pathologies genetics, cell biology or, as in my and target different areas of the own case, a link with head injury, a brain (Steve de Armond, Univer- large number of groups have come sity of California, San Francisco, to think of [3APP as being central USA). to unravelling the mechanisms of The meeting ended with an im- AD. This meeting provided a valupromptu session on the current able insight into what is now known state of transgenic animal re- about the processing and function search. The session opened with of ~IAPP and, as with all good Alison Goate (St Mary's Medical meetings, it raised just as many School, London, UK) giving a brief questions as it answered. I for one summary of the 717 mutations came away with a wealth of new identified in the [3APP gene. A ideas and a renewed enthusiasm to string of speakers then described get back into the laboratory.
Trends in Neurosciences
Subscriptions
All new subscriptions, renewals and subscription queries should be addressed to our Barking office at the address below.
[] Personal subscriptions (1992, 12 monthly issues): £59.00 in the UK; $92.00 in the USA; £63.00 elsewhere [] Library subscriptions (1992, 12 monthly issues plus hard-cover bound compendium with index): £239.00 in the UK and ROW [] Student subscriptions (1992, 12 monthly issues): £49.00 in the U K; $77.00 in the USA; £53.00 elsewhere (Student subscriptions must be accompanied by valid proof of student status.) All prices include postage (air delivery outside UK). All subscriptions can be paid for by cheque, bank draft or UNESCO coupons. Personal and Library subscriptions can also be paid for by credit card - Access/ Mastercard, Eurocard, Visa or American Express. Subscription orders should be sent to: Elsevier Science Publishers Ltd, Crown House, Linton Road, Barking, UK I G l l 8JU. Tel. (outside UK): +44 81 5947272. TeL On UK): 081 594 7272
TINS, VoL 15, No. 9, 1992
