MOl.ECl u"\R Ml:.DICI E 10DAT, OCIUBER 1<)97 Perspectives

Memories re made of this: the genetic basis of memory

Liz Fletcher

Total amnesia is rare, but we face an 'epidemic' of memory loss. At present there are around 18 million people worldwide with Alzheimer's disease, and this figure is predicted to double in the next 25 years. While traditional clinical and experimental studies have elucidated much about the basic processes of memory and learning, modern genetic techniques look set to unravel their molecular mechanics. Only time will tell whether this knowledge will yield preventive or curative therapy for memory loss.

PROGRE SIVE forgetfulne i. something that we all face a part of the normal ageing proces , but memory 10 is al 0 as ociated with var­ious di ea. estate. The e include orne pathological neurodegenerative condition., Korsakoff's yndrome in alcoholics, and Alzheimer's di -ease. A more acute 10 of memory i aL one con quence of brain damage through stroke and tumours. To date, the only drug available for the treatment of memory loss i. for patient. with Alzheimer's dis­ease ( ee Boxe. 1 and 2). The increased number~ of people developing Alzheimer' disease alone mean that re earch into the proce. ses under­lying normal memory and learning has never been more pre. sing.

Forming a theol'Y of memory As early as I 94, Ramon y Cajal uggested that u. e lead to the st rengtheni ng of synaptic connection and that thi might be the mechani-m of 'memory torage' . By the turn of the twentieth cen­tury, it was widely accepted that the cortex wa, the home of learning and memory although Karl Lashley, then Professor of P. ychology at Harvard University (Boston, MA, USA), failed in hi attempt to de­fine a ingle memory ' hot pot" . In 1949, the anadian p ychologU Donald lI ebb (a student of La hley ' ) was the first to develop a . pe­cific model of u e-dependent change ill ynaptic trength. Hebb pro­po, ed that, under certain condition, d namic change in synap e , termed ' neuronal plasticity '. could be induced. The. e changes were

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Perspectives

Box 1. Memory loss in Alzheimer's disease

The loss of short-term memory in the early stages of Alzheimer's dis­ease is accompanied by damage in the entorhinal cortex. In time, the progression of the neuropathology leads to a generalized loss of cogni­tive ability and personality; and, ultimately, the patient becomes psy­chotic. However, the patient with Alzheimer's disease retains memories from childhood and early adulthood, and can become ' trapped in the past '. One drug treatment tbat has recently become available to those suffering from Alzheimer 's disease is Aricept (developed by Eisai Ltd, Japan, and marketed by Pfizer), which blocks the breakdown of acetyl­choline at the synaptic junction to boost the failing levels of this neuro­transmitter in the earlier stages of the disease. Clinical trials have yielded mixed results: there is significant improvement in some pa­tients but little in others. Harry Cayton, director of the UK's Alzheimer 's Disease Society, thinks that Aricept is an important advance because although it does not delay, stop, or cure Alzheimer 's disease, it does suppress some of the symptoms and could offer an improved qual­ity of life for those in the earlier stages of the disease.

restricted to coincidentally act ive neurons, and had to be long la ting o tbat an amplified response could be evoked at the synapse at some

later date. In essence, Hebb had devised a theoretical model for memory; a potential physiological correlate emerged later, however, in tbe di covery of the phenomenon of long-term potentiation (LTP). In 1973, physiologists Tim Bliss and Terj e L0mo discovered that a short burst of electrical 'shocks' to afferent pathways in the hip­pocampus evoked an impressive, long- lasting amplificat ion of the

Box 2. Memory-enhancing agents

Nootropics are memory-enhancing agents that aim to improve concen­tration, memory retention and problem-solving ability; they are rapidly becoming big business, particularly in the USA. 'Genuine ' nootropics include the pyrrolidone derivatives piracetam and oxiracetam, which appear to work by enhancing the blood flow to the brain. Other candi­dates have diverse origins: these include deprenyl (the anti-Parkinson ' disease drug), phenytoin (an anti-epileptic) and the alkaloid vincamine (also a vasodilator). In nearly aU cases, the effects of these drugs are minimal (little more than the action of a strong cup of coffee) and the pharmacological basis of their action is poorly documented; some of them also have unpleasant - or even dangerous - side effects.

More recent 'wonder ' drugs have been christened ampakine by neuroscientists Gary Lynch and Charles Stevens (the Salk Institute, La Jolla, CA, USA). Aropakines boost ynaptic ignalling in the brain (and potentially memory processing) by enhancing the action of glutamate at the a-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMP A) subtype of receptor [related to the N-methyl-D-aspartate (NMDA) recep­tor, and also important for LTPJ. These drugs are currently being devel­oped by Cortex Pharmaceuticals (Irvine, CA, USA), and in preliminary tests have shown a 20% improvement in short-term recall and memory tests in healthy young male volunteers. However, it is unclear what ide effects the ampakine might produce or whether they will enhance dam­aged memory.

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MOLECULAR MEDI C I NE TODAY. OCTOBE R 1997

subsequent responses in those same pathways. By the 1970s, neuropsychologists Brenda Miller and Larry Squire already had evi­dence that the medial temporal lobe (including the hippocampus and amygdala) was involved in certain forms of memory in humans and in experimental animals. Hippocampal LTP thus seemed a possible candidate for one of the underlying physiological mechanisms.

LTP has remained a favoured neurological model for memory re­searchers over the past two decades. The phenomenon has also been recorded in structures other than the hippocampus, such as the neo­cortex. Refinements of experimental techniques, and the develop­ment of pharmaco logical agents for di ssecting out the contributing neurochemica l mechanisms, have significantly advanced our under­standing of LTP. However, this approach has still left many gaps in our understanding; hopefull y, some of these will now be addressed u ing molecular biology.

Types of memory There are two temporally distinct forms of memory: short-term mem­ory is of limited capacity and la ts for minutes or hours, whereas long-term memory is more elaborate and lasts for days, months, or even a lifet ime (Box 3). The processes underl ying these two forms of memory are pharmacologically distinct because short-term memory is disrupted by anaesthetics, while the formation of long-term memories is blocked by protein synthesis inhibitors. Researchers such as Eric Kandel (Columbia University, ew York, NY, USA) have long puz­zled over the molecular switch that converts the short-term process into a more durable one. The Californian marine snail Aplysia calif or­nica is particul arly amenable to the study of such a switch : if the siphon of Aplysia is prodded, it retracts its gills; if its tail is then shocked, it remembers the in ult , and minutes later will react more en­ergeticall y to a further prod. At the cellular level, this lea rning leads to an increase in synaptic strength between sensory and motor neurons: a form of short-term memory. After a period of ' rest', the snail forgets its rough treatment and the synaptic potentiation declines to pre-stim­ulu levels. If, however, the tail shock is repeated four or five times over a one-hour period, the snail reacts strongl y to the prod for days or weeks: a form of long-term memory. The trigger for this switch from short- to long-term ' imprinting' is what is now under investigation.

Serotonin and CREB Kandel's group has used miniature cellular networks consisting of a single ensory neuron synapsed to a single motor neuron to attempt to identi fy the chemica l switch(es) involved. Application of serotonin (a mod ulatory neurotransmitter released following the tail stimulus) to the co-culture can replicate the physical prod, and causes a measur­able rise in cyclic adenosine monophosphate (cAMP), which then ac­tivates a variety of enzymes capable of modifying the function of ex isting cellular proteins. With repeated erotonin applications (or prods), the cAMP level remai ns elevated for longer and provides the chemical witch that is critical for more permanent synaptic modifi­cation. Kandel and his colleagues have shown that cAMP signalling activates a tran cription factor, cAMP response element-binding pro­tein (CREB); which controls the expre sion of a variety of genes and would ex plain the dependence of long-term memory on protein yn­thesis. In this way, transient changes in electrical activity of the ce ll are converted into change in cellular metabolism.

Regulation of gene ex pression by CREB might be a common mechanism for the formation of memory in many species: disruption of the eREB gene al 0 disrupts long-term memory formation in

M O L E

mice. What is less clear is which genes are regulated by CREB acti­va tion, and what role they play in the creation of memory. A vast number of so-called immediate earl y genes such as c-Jos. C-jllll and ziJ/268 have been identified; defining their function, and the path­ways in which they operate, is therefore currentl y an area of intense research effort.

Tagging neurons for LTP Kandel believe that Aplysia is an excellent model with which to study ' the most wonderful problem in cell biology '. He question: if there is a single neuron with ten branches, and you give a short burst of high-frequency shocks to tetanize just one synapse, do you get strengthening at all ten branches or just the activated one? Kelsey Martin in Kandel's group has developed a system in which a single neuron with two bifurcating axons can be isolated. If they 'puff ' serotonin on one synapse alone, onl y the activated terminal gets stronger, even though transcription is initiated at the nucleus. Kandel adds, 'We'd now like to know how the terminal is "marked" for LTP.' Richard Morris (currently at the Centre for euroscience, University of Edinburgh, Edinburgh, UK) and Uwe Frey (Federal Institute of Neurobiology, Magdeburg, Germany) have already ad­dressed this issue in part , using the hippocampal LTP model. They found that they could induce long-lasting LTP (, late LTP ') at one set of synapses and, one hour later, induce late LTP at a second set of synapses on the same pyramidal ce lls, even though a drug that blocks protein synthesi had been applied. They believe that a ' tag' is gener­ated at the synapse at which LTP is induced. This tag can sequester proteins that are essential for stabilizing late LTP. Thus, it eems likely that proteins critical for LTP don't have an ' address ' on them -they simply diffuse through the ce ll until they are hijacked by a synaptic tag. Identifying the nature of the synaptic tag will be an im­portant breakthrough in our under tanding of the principle of neur­onal plasticity.

LTP: memory or not? While LTP has many attractions as a cellular model for memory and learning, researchers have had, until recentl y, onl y indirect ev idence that this model is valid. In 1986, Richard Morris tested the effects of an infusion of an NMDA receptor antagonist called 2-amino-5-pho -phonovaleric acid (AP5) directly into the brain of rats trained in the Morris water maze (a test of spatial memory). Administration of AP5 abolished LTP and also the rats' performance in the maze; the result wa enthusiastica ll y taken as ev idence for a role for LTP in memory in vivo. The behavioural effects of AP5 could have stemmed from blocking the function of the NMDA receptor elsewhere in the brain. However, infusions of AP5 directl y into the hippocampu produce the same effect.

Creating a forgetful mouse ew tran genic techniques prov ide a means to a sociate the function

of specific molecul es with memory by deleting . ingle gene. and studying the phy. iologica l and behavioural consequences in the living animal. The first two ' forgetful ' mice were created in 1992 by Alcino Silva (now at old Spring Harbor, Y, USA), Seth Grant (now at the

entre for Genome Re earch and Neurosciences, University of Edinburgh, UK) and their colleague. ilva ' team eliminated a ­Ca2+-ca lmodulin-dependent protein kina. e II (a aMKJI), an abun­dant enzyme in the hippocampu and onc known to be important in LTP. Thc aCaMKII knockouts performed poorl y in the Morris water

Perspectives

Box 3. Definitions of memory

Declarative memory This is episodic and involves memories of places, people and things. Good examples are the recollection of a friend 's face, a favourite paint­ing or where you left the car keys. The hippocampus is critical in its formation, although brain imaging tudies have implicated cortical re­gions in its storage and retrieval. In the laboratory, the 'Morris water maze' and 'context-dependent fear conditioning' provide test of de­clarative memory in rodents.

Procedural memory This is the form of memory that involves strategies and actions. Good examples include remembering how to drive a car, or the rules of gram­mar learned at school. It is not dependent on the hippocampus but might involve other brain regions such as the cerebellum. In the labora­tory, the sea snai l Ap/ysia and the fruit fly Drosophila are both amenable to studies of procedural memory.

maze, and LTP could not be induced in the hippocampu in viTro -evidence both for a role for aCaMKJI in memory-inducing pathways, and for a role for LTP in memory formation. A number of other elec­tive gene knockouts have prov ided a variety of memory-defective mice (see Table 1). While conclu ive evidence for a direct link be­tween LTP and memory has not yet been forthcoming. the general pattern is in agreement with the pharmacological studie , sugge ting a close association.

Problems with knockouts Although elective gene knockouts have undeniably been of great benefit to the memory-research community, they are not without their limitations. Knockout are generated through modification of stem cells, so the gene of intere t i eliminated from every cell in the animal at the ea rlie t stage of development. Thi might disrupt the normal development of the brain and , becau e genes frequentl y have multiple functions, might affect more than one ph ysiological proces . An example of thi is the 'global' MDA knockout mou e: the mice died at birth from respiratory fa ilure, highlighting the importance of

MDA receptors in proce e other than memory formation. A fur­ther complication i that other gene can compen ate fo r the elimi­nated gene, again making interpretation of the behavioural con e­quences of a single gene mutation far from traightforward .

Knockouts in time and place Several of the pitfalls of 'global' gene knockout have now been cir­cumvented by some ingenious new technique (ee Fig. I) that can generate cell -type-specific gene knockouts. One such technique is ' noxing', in which the gene of interest i selectively exci ed in a cell­type- pecific manner. Last year, Su umu Tonegawa and colleague (Center for Memory and Learning, MIT, Cambridge, MA, USA) re­ported that they had succe sfull y 'noxed out ' the MDA re eptor fro m the A 1 region of the hippocampus (an area in which LTP can be in­duced). Tonegawa's team in collaboration with Mark Mayford and Eric Kandel, placed Cre recombinase (a bacteriophage enzyme that ex­ci es D A between a pair of palindromic sequences ca lled 10xP ite ) under the arne promoter as a aMKJI , which i expres ed predomi­nantl y in A I cell. . They al 0 replaced the native MDA receptor

431

Pers pecti ves MOLECU LAR M E D I C I N E TODAY. O CTOB E R 1997

Table 1. Gene knockouts and their effects on memory Targeted gene product Function of gene product Effect on spatial memory Effect on LTP Notes Ref(s)

Ca2'-<:aImodulin-dependent Protein kinase implicated Impaired (site-directed No LTP in hippocampus (with a few exceptions)

Seizures in a protein kinase in leaming overexpression of the gene

also caused impairment) the mice; enhanced acoustic startle response

Protein kinase C-'Y Protein kinase involved in many signal transduction pathways and implicated in LTP

Mild deficits only Abnormal LTP but only induced by low-frequency stimulus

'Y isoform is specific to CNS and is expressed postnatally

b

cAMP-dependent protein kinase

Protein kinase involved in many signal transduction pathways

Impaired in some knockouts; impaired by overexpression of inhibitory subunit

No LTP in hippocampus No anatomical abnormalities

c

Thy-l

Fyn

mGLuR1

Adenylate cyclase

NMDAR1 (CAl only)

Neuronal glycoprotein Unaffected and cell adhesion molecule

Protein tyrosine kinase Impaired

A glutamate receptor implicated in L TP

Enzyme that catalyses

the production of cAMP

Essential subunit of the NMDA receptor

Impaired

Impaired

Impaired

L TP in dentate gyrus but not hippocampus

No LTP (unless high frequency stimulation is used)

Variable

Reduced

NoLTP

No anatomical d abnormalities

Defect in brain e cellular architecture

Contradictory results in two studies

g

Elimination of h NMDA expression in CAl region

"Silva, A.J. et II. (1992) SaiItIce 257, 201-206; Silva, A.J. fit aI. (1992) Science 257, 206-211 ; Mayford, M. et aI. (1 996) Science 274, 167S-1683 'AbeItovich, A. fit aI. (1993) CB/175, 1253-1262; AbeIiovich, A. et aI. (1993) CB/175, 1263-1271 'Brandon, E.P. fit 81. (1995) Proc. NBtI. Acad. Sci. U. S. A 92, 8851-as55; Qi, M. fit aI. (1996) Proc. foIalI. Acad. Sci. U. S. A 93, 1571-1 576; Abef, T. et aI. (1997) Ceil 88, 61~ 'Nosten Bertrand, M. et 81. (1 996) Natute 379, 826-829 'GrIr1t. S.G.N. fit 81. (1 992) ScietIce 256, 1!m-1910 'Conquet, F. et aI. (1994) Natute 372, 237- 243; Aiba, A. fit aI. (1994) Cell 79, 36S-375 fNu, Z.l fit 81. (1995) Proc. NatI. Acad. Sci. U. S. A 92, 220-224 "Tsien, J.l. fit aI. (1996) CetY 87, 1317- 1326

subunit NRI (critical for LTP induction) with a ver ion that was fl anked by 10xP sites. When the two mice were crossed, their progeny produced Cre recombina e specifically in CAl cell , which then re­moved the 10xP-fi anked NRI gene, yielding mice Ihat lacked NMDA receptors specifically in the CAl pyramidal cells. 0 LTP could be measured in the hippocampu in vitro, and the mice performed poorly in the Morris water maze, helping to confirm the link between hip­pocampal LTP and certain fo rms of memory. In collaboration with Matthew Wilson of M1T, the researchers further confi rmed a role for the hippocampus in spatial memory. Each mouse wa fitted with a tiny helmet of 30 electrodes that were lowered into the hippocampus. The electrical activity of Ih is tissue could be recorded online a the mouse

43 2

moved between locations. In conlrol mice, there wa an orga nized paltern of firing as Ihe mouse moved between pecific locations; in Ihe NMDA knockout , thi paltern was disrupted.

With a uilable promoter, the Cre recombinase sy tem could be used to eliminate any gene in any of a num ber of ce ll types. However, thi method of gene knockout is both irreversible and trig­gered ea rl y in development. To overcome these potential limitations of the technique, Mayford, Kandel and their coll eague have adapted a drug- inducible knockout system to investigate the role of aCa MKII in memory fo rmation in the normal nervous sy te rn . They placed the gene of interest (a constitutive ly acti ve form of aCaMKJI) under a tetracycl ine-sen iti ve control y'stem that prov ides

10xP site

aCaMKii promoter

\

Excision of NR1 gene

Mouse 1

CA1 neurons

o •

aCaMKii

Cross mouse 1 with mouse 2

Progeny

Mouse 2

0-All other cells

No excision of NR1 gene

Pers pecti ves

Cre recombinase enzyme

Figure 1. Floxing genes. A pair of palindromic sequences called 10xP sites is inserted around the gene of interest (in this case the N·methyl-D-aspartate receptor subunit NR1 ), ensuring that the expression and function of the gene is not disrupted. This 'floxed' (from flanked by 10xP) gene is then injected into mouse embryonic stem cells and will, with luck, recombine with the native gene, resulting in a mouse containing the floxed gene. In a second line of mice, the gene encoding Cre re­combinase is inserted into the genome under the control of a cell·specific promoter (such as the promoter for a·Ca2·-calmodulin-dependent protein kinase II , aCaMKII); the recombinase enzyme will thus only be expressed in a specified cell type. The two mouse lines are then crossed and a mouse containing both trans­genes is identified. In such mice, the Cre enzyme induces the excision of the floxed gene but only in those cells in which the enzyme is induced.

temporal control of gene ex pres ion. In another mouse line, they placed the tetracycline control ystem under a cell- pecific promoter (the one they had u ed earlier for native aCaMKII); this provide spati al control of gene expres ion. When the two train are cro ed, the off pring have a tetracycl ine- en itive aCaMKII tran gene pe­cific for the A 1 region of the hippocampus. In the absence of tetra­cycline the animal. howed memory deficit , confirming a role for thi. enzyme in memory formation. Conversely, in the presence of the drug and therefore, production of aCaMKII , the animal rega ined their ability to learn tasks. 'To my knowledge, controlled or inducible gene deletion ha not been achieved in the brain,' says Mayford. 'However, this is clearly one of the direction that the research i head ing. Modifications of the tetracycline ystem may be u eful in thi. endeavour.'

The futu re Refining the technique for gene targeting i also an important goa l of Kandel s team. 'We need to find beller promoters 0 that we can

get more discrete expression of gene in the brain,' ays Kandel. The problem they want to addre i what each region of the brain i doing in memory formation. ' Do different regions control different

Qutlllon. ariling for molecular medicine • What other forms of synaptic plasticity underlie memory and learning? • Where and bow are forms of memory, other than spatial memory, generated? • How do hormones and growth factors, which are also regula­tors of gene expression, influence memory formation? • Will it be possible to engineer transgenic animals to enhance, rather than elimiDate, memory? • How can this knowledge be used to design memory-enhanc­ing therapy or prevent memory loss?

433

Perspectives

a pect of memory, for example the CAl region for spatial memory, the hippocampu for memory of people and place , and the ento­rhinal cortex for olfactory memory, and so on')' The alternative is that all region might work 'with all sen ory modalities. but just special­ize in one particular a pect of memory encoding, torage or retrieval. If we can control gene expres ion more tightl y we could ystemati­cally interfere with LTP in each region and identify what ort of memory i disrupted.'

Finding a cau al link between pia tic change in brain synap e and the proce e underlying mammalian memory and learning no longer seem a formidable ta k. Reflecting on tbe fate of LTP in memory and learning, Richard Morris thinks it 'goe in and out of favour. ' He ays tbat he i . till a believer' and that he ha orne ex­citing new data (u ing APS) to ub tantiate hi hunch, He u pect that LTP is important for certain kinds of learning, but probably not as a general mechani m. Morri feels that a pharmacologica l ap­proach to investigating LTP mechani m continues to be attractive and that with orne really pecific drug. experiment can be per­formed that are still not po ible using knockout. To test the effect on memory directl y. Morris ays. 'We can now take an animal, can­nulate the hippocampu and put in a variety of drug : APS one day, aline the next, an AMPA antagoni t the next. .

With new tran genic technique . re~earcher can now examine the contribution made by specific genes to memory functions, Other in­ve tigator are attempting to under tand better the neuropathological basi for memory disruption in neurodegenerative condition such a ' Alzheimer ' disea e (Box 1); for example, recent tudies in a tran -genic model for lzbeimer' revealed a di ruption of LTP. Together,

M()L1,CULAf~ MFDICINE TODAY. O C rOBER 1997

molecular biologists. phys iologi ts and pharmacologists can start to build a picture of the molecule of memory, and so lay the foun­dation for potential treatment for amne ia.

Selected reading Bli , T.v.P. and CoUingridge, G,L. (1993) ynaptic model of mem-ory: long-term potentiation in the hippocampus, a/ure 37 , 31-39

Chen. 1. and Tonegawa, S. (1997) Molecular genetic analy i of ynaptic plasticity, activity-dependent neural development, learn-

ing, and memory in the mammalian brain, Anllll.,R ev. ellrosci. 20. 157-1 4

Frey, U, and Morris, R.G. M. (1997) ynaptic tagging and long-term potentiation, alLlre 38-, -33-536

Grant, S. and ilva, A.J. (1994) Targeting learning, Trends Neurosci,

17,71-75

Tsien, J.Z. e/ 01. (1996) ubregion- and cell type-restricted gene knockout in mouse brain, Cell 7, 1317-1326

T ien, 1.Z. e/ 01. (1996) The e sential role of hippocampal CAl MDA-receptor-dependent synaptic plasticity in patial memory,

Cell 7, 1327-133

Liz Fletcher is a freelance science writer.

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