Initiation and propagation of molecular cascades in human brain aging: Insight from the canine model to promote successful aging

Prog Neuro-Psychophannocol.

&Bid. F’sychiat. 2000, Vol. 24, pp. 777-786 Copyright

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SECTION II INITIATION AND PROPAGATION OF MOLECULAR CASCADES IN HUMAN BRAIN AGING: INSIGHT FROM THE CANINE MODEL TO PROMOTE SUCCESSFUL AGING ELIZABETH

HEAD, PHILLIP L. THORNTON, LIQI TONG AND CARL W. COTMAN

Institute for Brain Aging & Dementia University of California-h-vine Irvine, CA, USA

(Final form, June 2000)

Contents

1. 2. 3. 4. 5. 6. 7.

Abstract Introduction The Initial Stage: Early Events in a Pathological The Role of Aj3 in Neuron Dysfunction: In Vitro The Role of Ag in Neuron Dystiuiction: In Vivo Intervention During the Initiation Stage How Can the Dog Help Us Understand the Early Summary Acknowledgments References

Cascade Studies Studies Events in a Pathological

Cascade?

Abstract

Elizabeth Head, Phillip L. Thornton, Liqi Tong, and Carl W. Cotman: Initiation and Propagation of Molecular Cascades in Human Brain Aging: Insight from the Canine Model to Promote Successful Aging. Prog. Neuro-psychopharmacol. & Biol. Psychiat. 2000, & pp. 777-786. %2000 Elsevier Science Inc.

1. Normal aging is thought to proceed through two stages: initiation and propagation. Each of these phases is associated with different neuroanatomical events, vulnerabilities to injury and responsiveness to interventions, 2 The role of J3-amyloid (AJ3) in neuron dysfunction in the initiation stage may be mediated through alterations in signal transduction pathways involving cyclic AMP response element binding protein 777

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E. Head et al.

(CREB). CREB phosphorylation is associated with the expression of brain derived neurotrophic factor (BDNF), which promotes neuron health and survival. In primary neuronal cultures, Ag decreases the phosphorylation of CREB, which results in up to a 3 1% decrease in BDNF levels. 3. In vivo studies also support a role for AB in neuron dysfunction since soluble Ag levels correlate with the loss of synapses in brains of non-demented humans with high pathology. 4. The authors hypothesize that interventions during the initiation stage, when neuron dysfunction, but not overt pathology, is present, have the most promise to promote successful aging. The dog can serve as a useful model for interventions during the initiation stage since dogs develop neuropathology that closely resembles that observed in high pathology human brains.

Kevwords:

aging, P-amyloid, bax, bcl-2, canine model, CREB, BDNF, oxidative stress,

Abbreviations: g-amyloid (A@, Alzheimer’s disease (AD), brain derived neurotrophic factor (BDNF), calcium calmodulin-dependent kinase (CAM kinase), cyclic AMP response element (CRE), cyclic AMP response element binding protein (CREB), mild cognitive impairment (MCI), proliferating cell nuclear antigen (PCNA).

1. Introduction

We conceive of the changes that take place with aging in the brain as a series of stages, each stage associated with different vulnerabilities

and ability to cope with injury. In successful aging, when there is

very little indication of cellular dysfunction,

the system has the capacity to maintain neuron survival for a

hundred plus years. Obviously some humans age successfully impairment (MCI)(Petersen,

yet develop a form of mild cognitive

et ab, 1997; Petersen, et al., 1999). This relatively benign form of aging is

associated with some of the same events that occur in disorders associated with pathological as Alzheimer’s

disease (AD), but in successful aging, these processes

aging, such

appear to be slowed or halted.

hypothesis is that normal aging converts to pathological aging by proceeding

One

through two stages: the

initiation stage and thepropagation stage. The initiation stage can be influenced by relative risk factors (e.g. ApoE genotype) and the expression

of neuroprotective

factors (Connor, et al., 1997; Ferrer, et

al., 1999; Johnson, et al., 1998; Knusel, et al., 1996; Phillips, et al., 1991). This phase is relatively stable, reversible and amenable to interventions.

The propagation stage is distinct in that it is self-reinforcing

via

molecular cascades; these cascades supercede risk factors and accelerate pathogenesis.

Once the propagation

stage is initiated, the bulk of molecular studies indicate that there are cascades of

events that have positive feedback loops and drive the pathology in an insidious, unstoppable virtually a molecular avalanche.

fashion,

So the real challenge for science is to begin to understand the earlier

initiation phase and to intervene at this point, with the prediction that success of interventions initiation stage will be higher than those at the propagation

stage.

at this

Promoting successful

aging using the canine model of aging

2. The Initiation Stage: Earlv Events in a Patholoaical

779

Cascade

To study the initiation phase in vitro or in vivo, it is necessary to develop a model where subthreshold insults occur that do not cause overt cell death but rather impair cellular function. to promote cell dysfunction

is AP since this neurotoxic protein accumulates

An appropriate

stimulus

in the form of senile plaques

in the aged human and canine brain. Exposing cell cultures to sufficient levels of Ag causes cell death in neurons and glia (Koh, et al., 1990; Korotzer, et al., 1993; Loo, et al, 1993; Pike, et al., 1994; Watt, et al., 1994). Neurons die by initiating programmed

cell death pathways; the up-regulation

proteins or the down-regulation

proteins (Paradis, et al, 1996)To identify events

of anti-apoptotic

associated with the initiation phase, we are now using sub-threshold

of pro-apoptotic

levels of Ag that do not cause overt

neuronal death to determine the sequence of events that occur early in response to an injury. These events may be subtle indicators of neuron dysfunction

that develops prior to the classic forms of pathology found

in the aged brain, such as senile plaques, neurofibrillary to a potentially toxic stimulus, signal transduction leading to neuronal dysfunction.

This hypothesis

tangles and cell death. Once neurons are exposed

pathways are activated that initiate a cascade of events led us to examine the role of signal transduction

pathways in neurons as early mediators of neuron dysfunction

and subsequent

3. The Role of AD in Neuron D&unction:

One of the components

that is critical in signal transduction

death.

In Vitro Studies

is CREB (cyclic AMP response element

binding protein), a molecule that mediates a plethora of responses involving gene transcription.

Briefly,

the pathway leading to the activation of CREB starts with an increase in Ca’+ or CAMP, which leads to the activation of calcium calmodulin-dependent

(CAM) Kinase IV or CAMP-dependent

(protein kinase A). CAM Kinase IV or protein kinase A translocates CRBB, thereby activating this protein.

Once CREB is phosphorylated,

into the nucleus and phosphorylates it can bind to cyclic AMP response

element (CRE) in the promoter region of specific genes and increase transcription, RNA and protein levels.

protein kinase

One of these proteins is brain derived neurotrophic

leading to increased

factor (BDNP), which

promotes neuron survival and plasticity (Cellerino, et al., 1996; Galuske, ef al., 1996; Ma et al., et

al., 1998). As the actions of CREB become more elucidated, it is becoming apparent that CREB is functionally

important for neuroplasticity

(Ahn, et aZ.,1999; Bailey, et al, 1996; Glazewski, et al., 1999;

Schulz, et al., 1999; Segal, et al., 1998). Recent data suggest that transgenic mice that do not express CRBB or mice treated with antisense mRNA to CREB, show impaired long-term potentiation, physiological

mechanism

a

thought to underlie short-term memory (Glazewski, et aI., 1999; Schulz, et

al.. 1999). A recent study also indicates that brains of humans diagnosed with AD have decreased levels of phosphorylated

CREB (pCREB) (Yamamoto-Sasaki,

et ab, 1999) and it is hypothesized

that this decline

E. Head et al.

780

may have a role in memory decrements.

While a direct mechanism or causal effect of declines in pCBBB

have not been established in normal aging in humans or animals, it is hypothesized that short-term memory deficits that occur as mild cognitive impairment (MCI) in humans or as memory impairments in individual old dogs may be a consequence of neuronal dysfunction associated with decreased phosphorylation of CRBB signal transduction mechanisms.

This leads to the question of whether a similar series of events occur with sub-lethal exposures to AB as would be expected in the early initiation phase. Depolarization of cells, as would occur in vivo with LTP, induces the phosphorylation of CRBB. However, in the presence of subthreshold levels of AB, there is a significant reduction in pCRBB (phosphorylated CBEB) (Tong, et ul.,In preparation). One interpretation of these results is that transcriptional activation by COBB could be compromised in dysfunctional neurons prior to overt cell death. If the neuron can reverse dysfunction, such as a diminished COBB signaling pathway, or an AB insult, then it can be returned to a functional state. Otherwise, an apoptotic program , may be initiated and the neuron removed from the system entirely.

The next hypothesis then is - does the loss of pCRBB affect the encoding of proteins that are involved in neuroplasticity? The answer appears to be yes. As previously mentioned, COBB is involved with the transcription of BDNP. BDNP is involved with learning and memory mechanisms, encoding LTP, cell health and survival, and protection from injury. In our culture model system, Al3 decreases depolarizationmediated induction of BDNP transcription by 3 1% (Tong, et ul.,In preparation). Thus, subthreshold Ag exposure can lead to some remarkable changes in neuron function including decreased transcription of BDNP, a protein important for promoting neuron survival, in the absence of any overt pathological change.

4. The role of AB in Neuron Dvsfunction: In Vivo Studies

There is evidence from in vivo studies for a role of Ag in the development of neuronal dystinction. Aged dogs and nondemented humans with high levels of AB pathology are potential systems for observing early changes in neuron function that are associated with the initiation stages. A recent report using high pathology control brains suggests that soluble A/3 rather than insoluble Ag deposited in senile plaques is associated with neuronal dysfunction as measured by synapse loss (Lue, ef al., 1999). Neurotibrillary tangle formation was not as predictive of synapse loss as was A/3. However, the best test is to use a model system where tangles are absent, such as in the aged dog, and we are currently testing whether soluble Al3 predicts synapse loss in this model. If we consider that AB can impair signal

781

Promoting successful aging using the canine model of aging

transduction mechanisms, leading to the loss ofBDNF expression, then one possible consequence of Ag exposure may be neuron dysfunction and synapse loss.

5. Intervention during the Initiation Stane

What types of interventions could be implemented in the initiation stage of brain aging to promote successful aging? One possibility is to regulate the expression of BDNF, a downstream product of the CRRB signal transduction pathway. Several years ago, we began to examine the possibility that simple behavioral interventions, such as voluntary running, could promote neuron survival via increased expression of BDNF. The experimental design of these studies includes providing rats with access to a running wheel and allowing each animal to run voluntarily and recording the distance traveled and speed. Voluntary exercise increased BDNF mRNA in hippocampal areas after several hours (Oiiff, et al., 1998), or days (Neeper, et al., 1995; Neeper, et al., 1996). We also looked at the possible involvement of CRRBmediated signal transduction and found that pCREB increased in response to the voluntary running paradigm during a period of seven days, while total levels of CREB were unchanged (Shen, et al.,In preparation). Recent data also suggest that exercise improves memory in aged humans (Binder, et al., 1999; Grealy, et af., 1999; Williams, et al., 1997) but the cause of this improvement in cognition has not yet been determined. However, together, these data suggest that exercise can be a driving force on plasticity mechanisms by enhancing the activation of factors that promote transcription of genes involved with neuron mnction and ultimate survival.

Are there other interventions that increase the expression of BDNF and have functional consequences? Several studies have been examining this question; the work on environmental enrichment is particularly important (Kempermann, et al., 1997; Kempermann, et al., 1998). Three weeks of environmental enrichment significantly stimulated cell proliferation, BDNF expression and resistance to insults, and inhibited apoptotic cell death (Young, et cd, 1999). Proliferating cell nuclear antigen (PCNA) levels were increased and double stranded DNA breaks (TUNEL) were decreased in the enrichment group relative to controls, suggesting that neurogenesis occurred in response to environmental enrichment.

Furthermore,

rats in the enriched environment were resistant to kainate-induced seizures and neuron death in response to seizures was ameliorated (Young, et d, 1999). Finally, the expression levels of BDNP were higher in the enrichment group relative to controls. Upstream of BDNF expression, the authors also showed that environmental enrichment increased the expression of CREB and pCRRB, particularly in the proliferating zone of new neurons. To summarize, these studies indicate that environmental enrichment and exercise, relatively modest interventions, can promote successful aging by modifying brain health at the single neuron level.

782

E. Head et al. 6. How Can the Don Help Us Understand

the Early Events in a Pathological

Aged dogs are well suited for the study of the early, initiation stage of pathological accumulate Al3 in an age-dependent throughout

pattern

Thus, to some extent, we can predict the

presence of Al3 in a specific brain region based on the age of a dog. By definition,

tangles (Cummings,

aging. Dogs

manner and deposition follows a relatively consistent

several cortical regions (Head, et ul.,in press).

develop Alzheimer’s

Cascade?

aged dogs do not

disease since they lack one of the classic markers of neuropathology,

neurofibrillary

et al., 1993; Okuda, et al., 1994; Uchida, et al., 1992). Thus, the brains of aged dogs

closely resemble those of high pathology controls discussed previously.

The authors have approached

the study of canine brain aging by measuring a number of different

markers that may be involved with pathways leading to or indicating neuron dysfunction of dogs ranging in age from 4-15 years. We have been interested in the expression oxidative stress as well as pro-apoptotic

and anti-apoptotic

in a single group

of indicators of

proteins, particularly in relationship

to the

deposition of Al3 (Head, et al., 1999). Our question has been: what is the sequence of expression indicators of neuronal dysfunction?

Fig 1 illustrates that the expression

of each of these markers varies as

a fimction of age in the temporal cortex of dog. Oxidative stress, mediated by peroxynitrite, measured by levels of nitrotyrosine

immunoreactivity.

of these

was

As Fig 1 shows, there are 2 time periods when

high levels of oxidative stress are observed: one between the ages of 5 and 6 years and the other between the ages of 9 and 13 years. On the other hand, expression

of bcl-2, an anti-oxidative

protein, reaches maximal levels between the ages of 5 and 9 years. Expression

and anti-apoptotic

of bax, a pro-apoptotic

protein, peaks very late in the lifespan, at 15 years, Finally, Aj3 deposition in the temporal cortex begins around the age of 10 years and rises in an age-dependent

fashion.

These results suggest that some event

between the ages of 5 and 9 years, possibly oxidative stress, occurs prior to the deposition of AB, and stimulates the system to produce survival factors.

7. Summary

When we consider all of these issues, it is clear that it is important to start to combine pharmacological therapies with behavioral implemented

must be

during the initiation stage for maximal efficacy. In the case of the dog, we have been

gathering information efftcacious.

strategies to promote successtul aging. These interventions

regarding possible “windows” where therapeutic interventions

For example, if an intervention

to the dog reaching 9 years of age.

would be most

to slow or halt the deposition of Al3 is best implemented

prior

In addition, we now have insight into the endpoints of intervention

at

Promoting successful

aging using the canine model of aging

783

the molecular, neuronal, cortical circuit and behavioral systems levels. The aged dog is a promising

model for examining these types of manipulations, and by looking at interventions implemented in the initiation phase, when the system has enough reserve, we can determine if we can promote successful cognitive and brain aging.

Fig 1. A schematic illustrating the sequence of events in the expression of a number of markers of neuronal dysfunction in the aged canine brain, a model of the initiation phase of brain aging. Patterns of expression levels for individual markers were determined in separate experiments using the same sample of dogs. The curves represent relative levels of expression for each marker as being high or low and are plotted in one graph for comparison purposes. A synthesis of our current studies suggest that one marker of oxidative stress, nitrotyrosine, peaks at two time points between the ages of 5 and 6 years and between 9 and 13 years. Expression levels of bcl-2 reach maximal levels between the ages of 5 and 9 years but drops dramatically after 10 years of age, around the same time that Ag deposition occurs in this sample of dogs. Bax does not show increases in expression until approximately I5 years of age. The x-axis represents the lifespan of beagle dogs and the y-axis represents relative levels of each marker.

Supported by funding from NIA AG12694 and AG13411. The authors acknowledge the helpful comments from Drs. Hong Shen and Kate Ivins and Nicole Berchtold.

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Inquiries and reprint requests should be addressed to:

Dr. Elizabeth Head Institute for Brain Aging & Dementia 1226 Gillespie Neuroscience Research Facility University of California Irvine, CA, 926974540 Email: eheadkiluciedu