Live discussion Tuesday, May 16, 2000, from noon to 1PM EST.

Panelists: Peter Davies, Virginia Lee, Gunnar Gouras, Bruce Yankner, Steven Younkin, Huaxi Xu and Charlie Glabe.

View transcript of live discussion (May 16, 2000)

Background and Discussion by Gunnar Gouras

Recently, an increasing number of reports are suggesting that intracellular accumulation of Aß42 may play an important pathological role in AD. It is now established that the Aß42 form of ß-amyloid increases with all known familial AD mutations and that the first amyloid plaques are composed of Aß42, and not the more abundantly secreted Aß40. Cell biological studies are increasingly emphasizing the subcellular site of Aß production, and are finding that Aß42 is abundant intracellularly as compared with secreted, especially in neurons. Remarkably, it was reported from the laboratory of Virginia Lee that an insoluble pool of intracellular Aß42 increases dramatically within neuronal NT2 cells as a function of aging in culture (Skovronsky et al., 1998). Recently, Aß42 accumulation was reported both in neurons in the vicinity of plaques (Mochizuki A et al.) and in neurons of AD susceptible brain regions even prior to plaques or tangle pathology (Gouras GK et al., Am J Pathol 2000). The recent publication by Naslund J et al in JAMA showing increases in brain Aß with early cognitive dysfunction and even prior to plaques and tangles, support the proposal that soluble Aß and not plaques may initiate AD pathology. Two other recent Aß ELISA studies are supportive of this (Wang J et al, Exp Neurol 1999, 158:328-337; McLean CA et al., Ann Neurol 1999, 4:860-6)

These recent publications counter the prevailing hypothesis of AD pathogenesis (via aggregated extracellular Aß toxicity within plaques) and raise the question of whether neuronal Aß42 accumulation may play a direct role in causing neuronal dysfunction, neuronal death and dementia. The prevailing hypothesis based on the the landmark discovery from the laboratory of Bruce Yankner that Aß when added to neurons is neurotoxic, may therefore have to be modified. A central question now is whether intraneuronal Aß42 accumulation merely reflects increased production with resultant increased extracellular neurotoxicity or whether intraneuronal Aß42 can also directly damage neurons from within.

Arguments Potentially Supporting Intracellular Mechanism

• Marked increase of intracellular Aß42 with aging in culture of neuronal NT2 cells (Skovronsky et al)
• Presence of intraneuronal Aß42 in AD brain (Mochizuki A et al, 2000) and in AD vulnerable neurons even before plaque and tangle pathology (Gouras et al, 2000)
• Supraphysiological levels of Aß needed to cause extracellular Aß toxicity in experimental models
• mRNAs isolated from plaques are derived from neurons (Ginsberg et al. 1999).

The following could argue against Aß plaque toxicity (i.e. aggregated, Congophilic plaque) and support an intracellular scenario, but also an extracellular toxic mechanism via soluble prefibrillar or protofibrillar Aß.

• Behavioral and physiological changes in AD transgenics even prior to plaques (see references in Gouras et al., Am J Pathol)
• Inflammatory changes prior to plaques (Sheng et al J. Neurochem, 2000)
• Recent ELISA data implicating soluble Aß with AD disease progression (see Naslund J et al )
• Evidence in vitro of suppression of LTP and apoptosis in neurons treated with low-molecular weight derivatives of Aß and protofibrillar Aß (see Live Discussion)

Arguments Against Intracellular Mechanism

• Extracellular Aß cause neurotoxicity in vitro and in vivo.
• Transgenic mice develop plaques without obvious neuronal loss, which argues against a necessary role for Aß accumulation in cell bodies in plaque pathology.
• A recent transgenic model with a neuronal promoter for ßAPP causing extracellular Aß deposition in vasculature and brain parenchyma (Calhoun ME, et al, 1999)
• Studies have shown that neurons can internalize Aß; therefore intracellular Aß (and associated toxicity) may be derived from extracellular Aß (Bahr BA, et al, 1998)

Discussion Topics

1) Recent ELISA studies indicate that Aß increases do correlate with cognitive status and also indicate that Aß increases prior to plaque and tangle pathology. One study suggested that it is soluble Aß that especially correlates well with dementia. So the question is where is this Aß? A recent study suggests that the earliest Aß42 accumulation occurs within neurons. Given that evidence increasingly indicates that the especially important Aß42 can accumulate within neurons, should we not be focusing more on this pool of Aß?

2) A recent study on the Novartis transgenic mice indicates prominent vascular Aß40 deposition when ßAPP is driven by a neuronal promoter. Does this prove that extracellular Aß is key? But then why is vascular Aß less commonly Aß42 and AD appears linked more closely with Aß42?

3) Although we still do not have a good understanding on the normal role of ßAPP or even Aß, neurons clearly generate intracellular Aß under normal conditions. The laboratory of Konrad Beyreuther has proposed that Aß is important for normal transport of ßAPP along processes. May an alteration in a normal intraneuronal function of Aß play a role in AD?

4) Evidence increasingly indicates that not only secreted but also intracellular Aß increases with familial AD mutations. What may influence increases in intracellular Aß in most forms of AD?

Next Steps

1) Determine whether plaques can form within neurons at nerve terminals via intraneuronal accumulation.

2) Create cell and preferably animal models that can isolate the potential pathological role of intracellular versus extracellular Aß.

3) Will experimental therapies aimed at reducing plaque load cause cognitive improvements, and if not, could this be from continued intraneuronal toxicity?

4) If intracellular Aß accumulation precedes tau pathology, determine the intracellular events that lead to subsequent cell pathology.

5) Work out the physiological mechanisms that can drive the acceleration of intracellular Aß accumulation.

6) Determine where within neurons Aß42 accumulation preferentially occurs.

Comment by Sam Gandy, posted 15 May 2000

The idea that intracellular Aß injures neurons and causes dementia prior to the formation of extracellular deposits is not obviously reconcilable with other evidence indicating that "full-blown" structural pathology precedes dementia (Crystal H, Dickson D, Fuld P, Masur D, Scott R, Mehler M, Masdeu J, Kawas C, Aronson M, Wolfson L. Clinico-pathologic studies in dementia: nondemented subjects with pathologically confirmed Alzheimer’s disease. Neurology 1988; 38: 1682-1687. Abstract. ; Price JL, Morris JC. Tangles and plaques in nondemented aging and "preclinical" Alzheimer’s disease. Ann Neurol 1999; 45: 358-368. Abstract).

Comment from Bruce Yankner, posted 15 May 2000

I would like to comment on several of the points made by Dr. Gouras in the background to our discussion. The prevailing amyloid hypothesis of AD does not require that plaques be the major toxic Abeta component. Rather, extracellular aggregated Abeta is proposed to be the major toxic component, and toxicity can be mediated by extracellular Abeta prior to plaque formation. This hypothesis is consistent with in vitro and in vivo Ab toxicity studies from many labs which show that extracellular aggregated Ab, not plaques per se, is neurotoxic. The proposal that intracellular Ab is also toxic and contributes to the neurodegenerative process should be carefully considered. However, there is still not a single report that shows intracellular Abeta toxicity. Sam Gandy's point is important - the literature suggests that dementia occurs only when extracellular pathology is evident. This does not rule out a contribution of intracellular Abeta, but it suggests that such an effect would be unlikely to precede extracellular Ab deposition.

Several arguments were advanced to support an intracellular Abeta mechanism.

1. Recent reports show intraneuronal Ab42 in a cultured neuronal cell line, in AD brain and in AD-vulnerable neurons before plaques and tangles. The key issue is not whether Ab is present in neurons but whether it does anything when sequestered in an intracellular vesicular compartment. Many groups have found that intracellular Ab, including Ab42, is a minor component of total Ab production. We have detected intracellular Ab42 in human cortical neuronal cultures from normal and Down's syndrome specimens, but this is a minor component of total Ab, even in 2 month old cultures. More importantly, in AD brains, intracellular Ab42 appears to be a minor component of total Ab42 immunoreactivity.

2. It was noted that extracellular Ab toxicity requires "supraphysiological levels". The micromolar concentrations of aggregated Ab required for Ab toxicity in vitro is in the range of Ab levels in affected AD cortex (Selkoe et al., 1986). Moreover, Ab toxicity in vivo requires only a single plaque-equivalent dose (Geula et al., 1998). If Ab toxicity occurred in the physiological nanomolar range, we might all have AD.

3. The behavioral and physiological changes in APP-transgenic mice prior to the appearance of many plaques argues against a plaque-based mechanism. The behavioral studies in APP-transgenic mice have been difficult to interpret because they are very strain-dependent. I think it is safe to say that something is happening to the transgenic brain which is separable from plaque formation, as suggested by Karen Hsiao and Lennart Mucke. However, what it is and whether it is relevant to AD is unclear. It is certainly too early to conclude anything about its cause. My guess is that it may relate to pre-plaque accumulation of fibrillar Ab.

4. The recent study by Naslund et al., which correlates brain Ab levels with disease progression, has been taken as evidence implicating soluble Ab. However, this study does not provide any information about the physical or cellular state of Ab. The paper suggests that Ab levels can be elevated prior to the appearnace of plaques, but this Ab could be extracellular, intracellular, soluble, oligomeric or fibrillar.

My own view is that both extracellular and intracellular Ab may contribute to the neurodegenerative process. However, to seriously consider a role for intracellular Ab, we need a study that convincingly demonstrates intracellular Ab toxicity.

Reply from Gunnar Gouras, posted 16 May 2000

We all agree that "full-blown" structural pathology (plaques and tangles) precede and accompany dementia. What we are especially considering is whether there is any role for intraneuronal Aß in causing neuronal dysfunction early on in the AD disease process. I don't think that anyone is convinced that intraneuronal Aß has a clear and seperate neurotoxic effect, but rather some of us are intrigued by the possibility that there may be an early pathological role for intraneuronal Aß42. Dementia is preceded by probably at least a few years of initially subtle and then mild cognitive impairement (MCI), the pathological basis of which is not established.

I appreciate the instructive comments of Dr. Yankner, and would like to add (Re: his point 1) that a major reason for my interest in intracellular Aß is that I studied the same brains as Naslund et al. did in their ELISA study with immunohistochemistry, and observed the most prominent Aß as intraneuronal Aß42 within AD vulnerable neurons and not in the brain parenchyma. I do not know whether this is from Aß42 uptake (see Bahr BA et l., 1998) or from endogenous accumulation. I also wonder if he has tried to extract Aß42 from neuron cell lysate with formic acid (see Skovronsky et al., 1998) or another more stringent method. Lastly, there was one report published in Nature Medicine last year of neuronal degeneration in thepresence of intraneuronal Aß42 acumulation but not plaque formation in a FAD PS1 trangenic mouse strain (Chui et al., 1999).

Comment by Dominic Walsh and Dennis Selkoe. Posted 15 May 2000

From various studies examining the toxicity of A-beta, it is clear that random coil monomeric A-beta is not toxic whereas oligomeric aggregates (protofibrils, ADDLs or fibrils) are. Thus the critical issue with respect to Abtoxicity is its aggregation state. An ideal anti-aggregation therapy would prevent the production of the first toxic assembly of A-beta.

It has been widely assumed that A-beta aggregation is initiated extracellularly. However, we have demonstrated (data recently presented at the Society for Neuroscience in Miami) that SDS-stable oligomers of A-beta (primarily dimers) are first generated intraneuronally. This finding represents the first direct observation of the aggregation state of A-beta in human neurons (earlier studies on intraneuronal A-beta did not deal with aggregation state) and establishes that oligomerization is initiated within cells. Irrespective of whether intraneuronal oligomers are toxic per se or mediate their effect after export to the extracellular space, their site of origin is now known. Thus, it would be desirable to interfere with A-beta dimerization within neurons.