This is an excellent paper that describes a novel model for assaying the effects of intracellular β amyloid peptide (Aβ) in primary neurons. The motivation for these studies is the increasing realization that Aβ can accumulate intracellularly, and this intracellular Aβ could be responsible for the neuronal toxicity observed in Alzheimer's disease. These authors have used an adenovirus-based transfection system in combination with a doxycycline-driven expression system to express human Aβ1-42 in primary rat cortical cultures. Induction of Aβ in these cultures resulted in an induction of a stress-responsive HSP70 protein and apoptotic death specifically in neurons. Coexpression of HSP70 by cotransfection resulted in a significant protection of neurons against Aβ-induced death.

As is typical for novel experimental approaches, this work raises many interesting questions, all of which should be addressable in this system. For example: Does intracellular Aβ expression affect tau phosphorylation? Does HSP70 directly interact with Aβ, and does HSP70 influence the cellular...  Read more

This is an excellent paper that describes a novel model for assaying the effects of intracellular β amyloid peptide (Aβ) in primary neurons. The motivation for these studies is the increasing realization that Aβ can accumulate intracellularly, and this intracellular Aβ could be responsible for the neuronal toxicity observed in Alzheimer's disease. These authors have used an adenovirus-based transfection system in combination with a doxycycline-driven expression system to express human Aβ1-42 in primary rat cortical cultures. Induction of Aβ in these cultures resulted in an induction of a stress-responsive HSP70 protein and apoptotic death specifically in neurons. Coexpression of HSP70 by cotransfection resulted in a significant protection of neurons against Aβ-induced death.

As is typical for novel experimental approaches, this work raises many interesting questions, all of which should be addressable in this system. For example: Does intracellular Aβ expression affect tau phosphorylation? Does HSP70 directly interact with Aβ, and does HSP70 influence the cellular distribution or oligomerization state of Aβ? Do other chaperone proteins (e.g., endoplasmic reticulum-resident HSP70s, or cytoplasmic αB-crystallin) provide similar protection? Does intracellular Aβ influence the processing of endogenous APP? Would Aβ1-40 have similar toxic effects? The answers to these questions may have direct relevance to the mechanism of neurotoxicity in AD, as well as the general issue of why AD is an age-associated disease.

View all comments by Chris Link

Jin et al. in the American Journal of Pathology and Magrané et al. in J Neuroscience provide further support for a critical role of intraneuronal β-amyloid accumulation. Jin et al. provide compelling data indicating that in cultured neurons with chemically induced Niemann-Pick C (NPC)-like phenotype, APP CTFs and Aβ42 accumulate in endosomal compartments, as previously noted by the groups of Hartmann and Ihara. Remarkably, they observe that cerebellar Purkinje cells from NPC brain (the cerebellum is relatively spared in AD) accumulate predominantly APP CTFs in early endosomes, while AD-vulnerable hippocampal neurons accumulate predominantly Aβ42 in late endosomes. These findings support increasing data (see Ohno et al., 2004) that it is specifically Aβ rather than full-length APP or APP CTF changes that are especially important.

In agreement with previous studies by LaFerla and Leblanc, among others, Magrané et al. provide further support for the exquisite neurotoxicity of intracellular Aβ42 within cultured...  Read more

Jin et al. in the American Journal of Pathology and Magrané et al. in J Neuroscience provide further support for a critical role of intraneuronal β-amyloid accumulation. Jin et al. provide compelling data indicating that in cultured neurons with chemically induced Niemann-Pick C (NPC)-like phenotype, APP CTFs and Aβ42 accumulate in endosomal compartments, as previously noted by the groups of Hartmann and Ihara. Remarkably, they observe that cerebellar Purkinje cells from NPC brain (the cerebellum is relatively spared in AD) accumulate predominantly APP CTFs in early endosomes, while AD-vulnerable hippocampal neurons accumulate predominantly Aβ42 in late endosomes. These findings support increasing data (see Ohno et al., 2004) that it is specifically Aβ rather than full-length APP or APP CTF changes that are especially important.

In agreement with previous studies by LaFerla and Leblanc, among others, Magrané et al. provide further support for the exquisite neurotoxicity of intracellular Aβ42 within cultured neurons. Using an inducible adenoviral vector containing Aβ42, they observe marked apoptosis with the Aβ42 construct, but not with the lacZ, APP full-length, or the Aβ reverse peptide sequence, Aβ42-1. Moreover, they report that HSP70 is upregulated with intraneuronal Aβ42, in agreement with prior work in C. elegans (Fonte et al., 2002). Remarkably, HSP70 overexpression appears to reverse apoptosis induced by intracellular Aβ accumulation, similar to analogous chaperones inhibiting intracellular prions in, for example, yeast. For further background on the Niemann-Pick study and endosomal APP/Aβ, one can read the accompanying review by Ralph Nixon, who, with his colleagues, pioneered the study of endosomal changes early on in Alzheimer’s disease.

View all comments by Gunnar K. Gouras

The biological consequence of intracellular accumulation of Aβ peptide has recently attracted increased attention. The Aβ42 peptide is present in brains of individuals with AD and Down’s syndrome, in APP transgenic mice, and in aging monkeys [1-6]. The observation that intracellular Aβ42 accumulation precedes the appearance of extracellular senile plaques and intraneuronal neurofibrillary tangles [NFTs] raises the question: Does intracellular Aβ42 contribute to early neuronal dysfunction and neuronal loss in AD and Down’s syndrome? The study by Magrane et al., together with a previous report by LeBlanc’s group [7], provides evidence that intracellular expression of Aβ42 is toxic to neurons cultured in vitro.

In this study, Magrane et al. constructed a tetracycline-regulated adenoviral system to express Aβ42 [8]. When Aβ42 was expressed in the secretory pathway of rat primary cortical neuron cultures, it remained inside the cell instead of being secreted into the media. Toxicity was detected in transfected neurons by TUNEL assay and by observing nuclear pyknosis. Strong...  Read more

The biological consequence of intracellular accumulation of Aβ peptide has recently attracted increased attention. The Aβ42 peptide is present in brains of individuals with AD and Down’s syndrome, in APP transgenic mice, and in aging monkeys [1-6]. The observation that intracellular Aβ42 accumulation precedes the appearance of extracellular senile plaques and intraneuronal neurofibrillary tangles [NFTs] raises the question: Does intracellular Aβ42 contribute to early neuronal dysfunction and neuronal loss in AD and Down’s syndrome? The study by Magrane et al., together with a previous report by LeBlanc’s group [7], provides evidence that intracellular expression of Aβ42 is toxic to neurons cultured in vitro.

In this study, Magrane et al. constructed a tetracycline-regulated adenoviral system to express Aβ42 [8]. When Aβ42 was expressed in the secretory pathway of rat primary cortical neuron cultures, it remained inside the cell instead of being secreted into the media. Toxicity was detected in transfected neurons by TUNEL assay and by observing nuclear pyknosis. Strong dosage-dependence on Aβ42 gives evidence that the toxicity is indeed induced by intracellular expression of Aβ42. In addition, Magrane et al. found increased Hsp70 levels in cells expressing intracellular Aβ42, indicating an induction of a stress response. Immunofluorescence data identified that the induction of Hsp70 expression is limited to Aβ42-expressing neurons. To test whether increased levels of Hsp70 is a cell’s attempt to rescue itself from intracellular Aβ;42-induced toxicity, Magrane et al. used an adenoviral construct to overexpress Hsp70 in primary cultured neurons. Intriguingly, overexpression of Hsp70 completely inhibited the neurotoxicity induced by intracellular expression of Aβ42.

Aβ42 used in this study contains a signal peptide from APP751 that directs it cotranslationally entering the ER. Using EM analysis, Aβ42 seems localized to the ER or is associated with vesicular membranes in these neurons. Hsp70 is a cytosolic chaperone, raising the question of how a cytosolic chaperone can inhibit the neurotoxicity induced by Aβ42 in the secretory pathway. Two scenarios can be speculated about regarding the mechanism of Hsp70 inhibition of neurotoxicity. First, increased levels of Hsp70 may suppress certain steps in the cell death pathway initiated by intracellular Aβ42 expression. Alternatively, as Magrane et al. suggest in the discussion, the ER quality control system could recognize Aβ42 as a misfolded protein. If Aβ42 were recognized as such, it would be retro-translocated to the cytosol. In this case, Hsp70 may directly bind to toxic Aβ42 peptide and inhibit its interaction with cellular partners.

The following reasons make the second possibility more plausible. First, although the majority of cells express Aβ42 (~70 percent transduction efficiency at 50 moi, or multiplicity of infection [8]), only 10 percent exhibit neurotoxicity. This discrepancy may suggest the requirement of further processing of Aβ42 in the secretory pathway for neurotoxicity. Retro-translocation of Aβ42 to the cytosol could provide this additional processing. Secondly, a previous study by LeBlanc’s group used microinjection to deliver Aβ peptide directly to the cytosol of human primary cultured neurons. They found that an extremely low level of Aβ42, 2.5x10-19 moles, is sufficient to induce rapid neuronal death [7]. Thirdly, it has been reported that Aβ peptide can inhibit proteasomal activity [9]. Thus, a small amount of Aβ42 could escape proteasomal degradation and induce toxicity. In addition, induction of the stress response in the cytosol could result from proteasome inhibition by Aβ42.

Further study is needed to demonstrate whether Aβ42 must be translocated to the cytosol to induce toxicity. The most direct evidence would come from detection of Aβ42 in the cytosol of dying neurons. However, detection of Aβ42 in the cytosol will be difficult to achieve, since a vanishingly small amount of Aβ42 in the cytosol is sufficient to induce rapid neuronal death. Demonstration of Hsp70 binding to Aβ42 would provide indirect evidence. However, such high amounts of Hsp70 are required to inhibit toxicity, as demonstrated in this study, that the binding between Hsp70 and Aβ42, if it exists, will be very weak and be difficult to detect. Alternatively, since cytosolic Aβ42 induces neurotoxicity through p53 and Bax [7], demonstrating that the toxicity induced by Aβ42 in the secretory pathway utilizes this same pathway would support this hypothesis.

These studies provide strong evidence for the neurotoxic effect by intracellular Aβ42. However, whether it is also neurotoxic in vivo or whether it contributes to the pathogenesis of AD remains unclear. The discovery of Hsp70’s inhibitory effect on intracellular Aβ42-induced neurotoxicity offers an avenue to test in animal models. However, overexpressing Hsp70 has so many different biological effects that a positive result from an animal study will not directly prove that intracellular Aβ42 contributes to the toxicity in AD in vivo. The in-vitro systems established in these two studies will allow us to dissect the neurotoxic pathway caused by intracellular Aβ42. Identifying specific inhibitors of this neurotoxic pathway and investigating their effects on AD animal models will ultimately lead to our understanding of the biological consequence of intracellular Aβ42.

References:
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8. Magrane J, Smith RC, Walsh K, Querfurth HW. Heat shock protein 70 participates in the neuroprotective response to intracellularly expressed beta-amyloid in neurons. J Neurosci. 2004 Feb 18;24(7):1700-6. Abstract

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View all comments by Jiyan Ma