Once in a great many experiments, a negative control can turn up surprisingly positive results. That happened to Todd Golde, Jungsu Kim, Terrone Rosenberry, and colleagues at the Mayo Clinic in Jacksonville, Florida, recently, when they identified an unexpected anti-amyloid activity in a peptide fragment of the Bri2 protein. The result is even more intriguing, given that mutated forms of the Bri2 peptide are themselves amyloidogenic and cause familial British and Danish dementias.

The work, published in the June 4 issue of the Journal of Neuroscience, puts the Bri2-23 peptide, comprising 23 C-terminal amino acids liberated from the parent protein by furin and related proteases, in a group with several other recently identified anti-amyloid peptides, including transthyretin (see ARF related news story) and cystatin C (see ARF related news story).

In a previous study, first author Kim used a Bri2-Aβ40 fusion protein to make transgenic mice that accumulated Aβ40 after furin cleavage of the fusion protein. Crossing those mice with Tg2576 AD mice revealed that elevating Aβ40 reduced amyloid buildup (see ARF related news story). That data led to the idea that Aβ40 was “anti-amyloidogenic” and protected against the aggregation of Aβ42. The new study replicates that finding in another AD mouse model (TgCRND8, expressing human APP with Swedish and Indiana mutations) using an adeno-associated virus (AAV) technology (“somatic brain transgenesis”) to introduce the fusion protein. Injection of the adeno-associated virus (AAV) vector into the cerebral ventricles of newborn mice results in robust expression of the fusion protein in the brain, and a reduction in total amyloid load and Aβ plaque load.

The surprise result came when Kim transduced mice with the full-length Bri2 protein as a control, and found that the Bri construct was as effective at reducing total amyloid load as Aβ40.

There have been previous reports that Bri2 interacts with APP and inhibits its processing in vitro (Matsuda et al., 2005; Fotinopoulou et al., 2005), but Kim and colleagues found no evidence for changes in APP processing in vivo, or for induction of a plaque-clearing immune response in the transduced mice.

Instead, they found that the Bri2-23 peptide could directly inhibit Aβ aggregation in vitro. In vivo, a mutant Bri2 protein lacking the C-terminal 23 amino acids had no effect on Aβ deposition. “Our data support anti-aggregation effects of peptides released from Bri2 as the mediator of anti-amyloid effects,” Golde told ARF. The results don’t rule out the possibility that Bri might under some circumstances affect APP processing, but either way, he said, “Bri2 looks like it is going to be a modulator of Aβ deposition.” If someone can find a way to deliver the peptide to the brain, Golde said, “We may have something that can be effective in slowing the course of amyloid deposition.”

Like transthyretin and cystatin C, mutant Bri2 peptides have the potential to self-aggregate and also interact with Aβ. “There may be a general theme emerging,” Golde said, “that if you allow two proteins that are potentially amyloidogenic or just in some way ‘unfolded’ to interact, maybe under some circumstances they may inhibit each others’ deposition.”

Could Bri2-23 be an endogenous binding partner for Aβ? In support of this idea, Kim and colleagues detected Bri2-23 in cerebral spinal fluid samples from three out of three people tested. There is also some unpublished genetic evidence that may point to a role for Bri2 in AD, says Golde.

Beyond the Bri2 results, the new study highlights the value of “somatic brain transgenics,” an approach Golde and colleagues have championed (see ARF related news story). Their technique of AAV-mediated gene transduction into newborn mice allows the rapid evaluation of transgenes in AD models at a relatively low cost. “A complete study with one construct in one or even two APP transgenic mouse models takes three to five months and costs $5,000 to $10,000,” Golde said. “That’s a fraction of the time and expense of making transgenics.”

Although Bri2 is highly abundant in the brain, the function of the normal protein is unknown, as is the basis for the neurotoxicity of mutated Bri2. A new transgenic mouse could be a start to understanding the pathology of the mutation. Ruben Vidal, Bernardino Ghetti, and colleagues at the Indiana University School of Medicine in Indianapolis have created a mouse expressing the Danish mutant form of Bri2 under the control of the mouse prion promoter, which they describe in a publication in Brain Pathology that appeared online in April (see also comment below). In the new mice, just as in human familial Danish dementia, Bri2 gets cleaved to yield the 34 amino acid, amyloidogenic ADan peptide, and similarly to the human disease, the animals show cerebral amyloid angiopathy, parenchymal ADan deposits, and neuroinflammation. Despite a large amount of CAA, there is no evidence of hemorrhages. The mice have abnormal grooming behavior, and gait changes. Oligomeric ADan (detected with the A11 antibody, which also recognizes oligomeric Aβ) is apparent in both intracellular and extracellular locations, along with tau immunoreactive deposits. Further studies on the mice may shed light on the question of whether amyloid peptides of different primary sequence share common pathogenic mechanisms.—Pat McCaffrey

Comments

Make a Comment

To make a comment you must login or register.

Comments on News and Primary Papers

  1. This paper shows the generation of a novel model of cerebral (non-Aβ) amyloid deposition. The authors generated transgenic mice expressing a mutant form of the BRI gene, found in patients affected by familial Danish dementia (FDD). FDD is a rare inherited disease that causes progressive dementia that, like AD, is neuropathologically characterized by amyloid deposition (ADan), neurofibrillary tangle formation (identical to that seen in AD), and neuronal cell loss. This model provides an exciting new tool in which to study the abnormal changes in the brain that lead to dementia. Comparing the similarities and differences of these two related neurological diseases may provide important clues to how AD develops.

    View all comments by Nikolaos K. Robakis
  2. This is a beautiful paper from Dr. Golde's lab showing for the first time that a peptide derived from the BRI2 gene is able to reduce cerebral Aβ deposition in vivo in an AD mouse model and that the same peptide inhibits Aβ aggregation in vitro. The peptide is a 23 amino acid long (Bri2-23) C-terminal fragment generated by the pro-protein convertases processing (Kim et al., 1999) of BRI2, a 266-amino-acid, type-II single transmembrane domain protein (Vidal et al., 1999). Using recombinant adeno-associated virus 1 (rAAV1)-mediated gene transfer in TgCRND8 mice, the investigators show a dramatic suppressive effect of the BRI2 transgene—and a BRI2-Aβ1–40 fusion protein (Kim et al., 2007)—on parenchymal Aβ accumulation. Importantly, the investigators found no evidence for alterations in the steady-state levels of APP or APP CTFβ in TgCRND8 mice expressing the virally delivered BRI2-Aβ1–40 or BRI2 transgenes, but rather that the Bri2–23 peptide could directly inhibit Aβ1–42 fibrillogenesis in vitro.

    Mutations in the BRI2 gene cause neurodegenerative diseases characterized by cerebral amyloid deposition (Vidal et al., 1999, 2000), and transgenic mice overexpressing a mutant form of BRI2 show cerebral amyloid (ADan) deposition (Vidal et al., 2008). Interestingly, the amino-termini of the amyloid peptides (ABri and ADan) contain the amino acid sequence of the anti-amyloidogenic peptide Bri2-23. The unexpected findings of Kim et al. generate even more questions regarding the normal role of the still poorly characterized BRI2 gene and how mutations in BRI2 lead to neurodegeneration. More work is needed to determine whether the Bri2-23 peptide is able to depolymerize mature Aβ fibrils and if the anti-amyloidogenic properties of Bri2-23 are also shared by the C-terminal peptides generated by other members of the BRI gene family (Vidal et al., 2001). The use of increasing levels of BRI2 in the brain for the treatment of AD as proposed by Kim and collaborators (Kim et al., 2008) is an interesting idea; however, we believe that since the normal function of BRI2 (and the Bri2-23 peptide) is not known, caution should be taken in attempting therapies based on the overexpression of BRI2 alone.

    References:

    . Furin mediates enhanced production of fibrillogenic ABri peptides in familial British dementia. Nat Neurosci. 1999 Nov;2(11):984-8. PubMed.

    . Abeta40 inhibits amyloid deposition in vivo. J Neurosci. 2007 Jan 17;27(3):627-33. PubMed.

    . A stop-codon mutation in the BRI gene associated with familial British dementia. Nature. 1999 Jun 24;399(6738):776-81. PubMed.

    . A decamer duplication in the 3' region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred. Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4920-5. PubMed.

    . Sequence, genomic structure and tissue expression of Human BRI3, a member of the BRI gene family. Gene. 2001 Mar 21;266(1-2):95-102. PubMed.

    . Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2. Brain Pathol. 2009 Jan;19(1):58-68. PubMed.

    View all comments by Bernardino Ghetti
  3. There are between 50 and 100 experimental manipulations that have been shown to alter the pathologic and/or behavioral phenotypes of various transgenic models of human Alzheimer disease. The description in this paper of the effect of the Bri protein, the agent of familial British dementia, by Todd Golde and his colleagues, is the latest example in which overexpressing a transgene encoding a wild-type protein in TgCRND8 model AD mice has an ameliorative effect on the AD phenotype. These observations are quite striking in the context of three other instances in which the expressed protein suppressing the AD phenotype is a precursor of a protein in which the wild-type or a mutant form is the proximal cause of human CNS or systemic amyloidosis. Similar effects have been found for cystatin C in Aβ Tg2576 (Mi et al., 2007) or APP23 (Kaeser et al., 2007) double transgenics; animals in which gelsolin, the precursor in the Finnish form of familial amyloidotic polyneuropathy (Hirko et al., 2007), has been expressed in Tg2576 and APP695/mutantPS1 mice transgenic for Aβ, and our own work describing the profound effect of overexpressing a transgene encoding wild-type human transthyretin in the APP23 model of AD (Buxbaum et al., 2008).

    Why should these proteins in particular have such an effect? If we assume that the excessive generation of Aβ1-42, its misfolding and subsequent aggregation into toxic oligomers and fibrils, is intrinsic to AD (as represented by these models), there are a variety of possible mechanisms that could explain the results. The overexpressed amyloid precursors may have a direct interaction with the Aβ fragment or its oligomers in the brain to either disaggregate them or accelerate their aggregation into larger non-toxic multimers that can be more rapidly engulfed and degraded by glia. They may bind to some factor that is critical for the generation of Aβ or its aggregation, reducing the concentration of fibrillogenic precursor. They may interfere with a downstream process responsible for neurotoxicity, having no impact on aggregation per se but a strong effect on the behavioral phenotype.

    In the gelsolin instance, the gene was introduced by hydrodynamic gene delivery and appeared to only be expressed in the periphery, not in the brain. Hence, its effect is hypothesized to be based on its action as a “plasma sink” for Aβ, increasing its transport from the brain to the systemic circulation, thereby decreasing the effective intracerebral Aβ concentration. A similar notion involving the CSF compartment has previously been proposed for the transthyretin effect. We think this unlikely (see below).

    The observations could be trivial since it is also possible that the effects may be mouse specific and have no relationship to human disease. Equally unlikely is the possibility that the apparent proclivity of this set of proteins to have the observed effect may represent a strong ascertainment bias in which the proteins in question are only a small sample of the universe of proteins that can do this, and the molecules that have been assayed for this property have been chosen precisely because they are amyloid precursors. For the purposes of the rest of my discussion I will ignore the last two possibilities and assume that the observations in the double transgenics and the gelsolin animals have some biologic relevance.

    Transthyretin, cystatin C, and gelsolin have been found in Aβ deposits in human AD brains. It has also been shown that in vitro the proteins directly interact with some form of Aβ, in the case of transthyretin most likely a subfibrillar aggregate. These proteins are apparently protective. We believe that their intrinsic amyloidogenicity indicates that they are predisposed to transiently expose their internal hydrophobic sequences to the external (with respect to the protein’s structure) aqueous milieu. If this occurs for a prolonged period or in a substantial portion of their conformational ensemble—conditions more likely for mutant forms of the proteins—the molecules will self-aggregate. However, if the molecule interacts with the hydrophobic portion of another similarly predisposed protein, the interaction can create a hydrophobic micro-environment for that protein domain. If the time frame is short enough, the remaining portions of the two interacting molecules re-fold to re-submerge the hydrophobic region into the internal portion of the native folded molecule. This process most resembles domain swapping but involves regions smaller than full domains and is temporally much more transient. Thus, there could be a series of proteins that are capable of protectively interacting with Aβ or its pre-toxic aggregates serving as “amateur” or “non-professional” chaperones for this particular cargo molecule.

    Why should such a mechanism be necessary? The relative frequency of neurodegenerative disorders related to gain of toxic function by misfolded proteins suggests that the usual proteostatic mechanisms operating in neurons are limited. The relative hypersensitivity of neurons to hyperthermia is consistent with this view. It is apparent that during the evolution of the central nervous system, selection has favored the production of limited amounts of functional small peptides. These, because of their size, are less likely to misfold, and are secreted in vesicles that are at neuronal termini, thus not exposing the rest of the cellular milieu to high concentrations of potentially misfolded molecules.

    These mechanisms serve the neuron well under most circumstances, unless there are destabilizing mutations in intrinsic neuronal proteins (e.g., α-synuclein, Huntingtin, SOD1). They may also fail when there is an interaction with an infectious agent capable of re-templating the folding of an endogenous protein. The system itself may become less effective (as in aging) for as yet unknown reasons. Under such circumstances, other mechanisms, such as those employing the “amateurs,” are recruited to cope. It is noteworthy that the transcription of transthyretin in the brain has been seen to increase in transgenic AD models. Interestingly, the AD models all require some degree of overexpression of the mutant Aβ construct, suggesting that the intrinsic murine neuronal proteostatic system functions well until it is overloaded. Old mice do not have an AD equivalent in the absence of overexpression of a human AD gene.

    It is also possible that the amyloidogenic proteins are not truly “non-professionals” but represent previously unrecognized elements of the neuronal chaperome. Richard Morimoto’s work in C. elegans is consistent with such a hypothesis in that mutations in known elements of the proteostatic machinery reduce the number of glutamines required to produce a neuropathologic phenotype in a poly-Q model of Huntington’s disease, but the effects of such mutations are not seen until the system is stressed, for example, by a misfolded protein challenge [see Bar Harbor Report 2007]. More broadly, cellular proteostasis networks and their role in health and disease are elegantly reviewed in Balch et al., 2008.

    Can these notions be experimentally tested for the proteins discussed here? Each observation should be validated by silencing the gene in question. Thus far, only deletion of the transthyretin gene has been tested for its effect on the development of a model of human Aβ transgene-induced murine AD. It accelerated the development of Aβ deposits in two different transgenic models, displaying a gene dose effect strongly supporting the notion that the observations were biologically relevant. If homozygous silencing of the gene in question is lethal, the effect of hemizygous silencing or siRNA knockdown of the gene on amplifying the Aβ phenotype should be reproduced as independent validation of the effect of the particular protein in question.

    The protein should be tested for its ability to bind to Aβ in vitro by some standard assay of protein interaction, and the nature of the molecular species of both the “chaperone” protein and Aβ involved in the binding should be defined. The protein should quantitatively inhibit the cytotoxicity of Aβ to neuronally derived targets at concentrations consistent with those attainable in vivo. Most difficult, but certainly most definitive, would be the demonstration of complexes between the protein and Aβ isolated from the target tissue of animals expressing both transgenes and controls.

    It would be desirable to determine whether introduction of the gene encoding the protein of interest somewhere in the course of the disease, rather than from conception, would have an impact on the development of the AD phenotype, suggesting that there might be some elements of these interactions that could be therapeutically exploitable. While it is conceivable that the observations made with respect to these four amyloid precursors are the result of ascertainment bias, until such bias is demonstrated the limits of the phenomena should be precisely defined and the underlying chemistry and biology thoroughly explored to determine if there is any “there” there.

    References:

    . Cystatin C inhibits amyloid-beta deposition in Alzheimer's disease mouse models. Nat Genet. 2007 Dec;39(12):1440-2. PubMed.

    . Cystatin C modulates cerebral beta-amyloidosis. Nat Genet. 2007 Dec;39(12):1437-9. PubMed.

    . Peripheral transgene expression of plasma gelsolin reduces amyloid in transgenic mouse models of Alzheimer's disease. Mol Ther. 2007 Sep;15(9):1623-9. PubMed.

    . Transthyretin protects Alzheimer's mice from the behavioral and biochemical effects of Abeta toxicity. Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2681-6. PubMed.

    . Adapting proteostasis for disease intervention. Science. 2008 Feb 15;319(5865):916-9. PubMed.

    View all comments by Joel Buxbaum

References

News Citations

  1. Fighting Fire With Fire—Transthyretin Therapy for Aβ?
  2. Genetic Risk Explained: Cystatin C Staves Off Plaque Formation in Mice
  3. Aβ40—The Anti-Amyloid?
  4. SfN Satellite Symposium: Neurotoxic Mechanisms in Alzheimer Disease

Paper Citations

  1. . The familial dementia BRI2 gene binds the Alzheimer gene amyloid-beta precursor protein and inhibits amyloid-beta production. J Biol Chem. 2005 Aug 12;280(32):28912-6. PubMed.
  2. . BRI2 interacts with amyloid precursor protein (APP) and regulates amyloid beta (Abeta) production. J Biol Chem. 2005 Sep 2;280(35):30768-72. PubMed.

Further Reading

Primary Papers

  1. . BRI2 (ITM2b) inhibits Abeta deposition in vivo. J Neurosci. 2008 Jun 4;28(23):6030-6. PubMed.
  2. . Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2. Brain Pathol. 2009 Jan;19(1):58-68. PubMed.