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The remarkable properties of amyloid-b derived from human Alzheimer’s disease brain: swinging the streetlight This scientific commentary refers to ‘Highly potent soluble amyloid-b seeds in human Alzheimer brain but not cerebrospinal fluid’ by Fritschi et al. (doi: 10.1093/brain/awu255). Despite many years of intensive study, the specific amyloid-b species in the human brain responsible for the pathophysiological processes underlying Alzheimer’s disease have yet to be identified. In part, this may be because we have been ‘searching under the streetlight’: examination of other sources of amyloid-b such as synthetic preparations, material derived from the brains of trans- genic animals, and amyloid-b recovered from human CSF after lumbar puncture has been much more convenient than direct evaluation of human brain-derived material. However, telltale signs over the last several years have indicated that amyloid-b derived from human brain may have properties or constituents that are qualitatively and quantitatively different from those of amyloid-b from other sources. The paper by Fritschi et al. (2014) from Mathias Jucker’s group in this issue of Brain is a major contribution to this line of investigation. Notably, the paper provides perhaps the most definitive evidence yet that human brain-derived amyloid-b has fundamentally dissimilar prop- erties to human CSF-derived material. Specifically, Fritschi et al. demonstrate that 5 1 attomole (10 18 moles) of water-soluble amyloid-b from frozen human Alzheimer’s disease brain tissue is sufficient to induce accelerated amyloid-b plaque deposition when injected into the brains of young APP transgenic mice. In contrast, human CSF from patients with de- mentia of the Alzheimer’s type containing over 100 000 times greater total mass of amyloid-b failed to affect amyloid-deposition when injected in the same manner. Neither intrinsic anti-aggrega- tion activity in CSF, nor freeze-thaw effects could explain these results; and CSF from transgenic mice behaved similarly, excluding any effect of species. The same lab has previously reported that synthetic amyloid-b at 100–1000-fold higher concentrations also fails to induce plaque deposition (Meyer-Luehmann et al., 2006). Furthermore, neither synthetic amyloid-b dimers nor protofibrils, mixed synthetic amyloid-b plus astrocyte-derived apolipoprotein E particles, nor cell-culture derived amyloid-b immunoreactive spe- cies induce in vivo plaque deposition (Meyer-Luehmann et al., 2006). The explanation for these remarkable observations is not at all clear. By way of tantalizing hints, Fritschi et al. reveal that the amyloid-b immunoreactive particles are larger in the human Alzheimer’s disease brain lysates than in the CSF, and their in vitro particle seeding activity is higher. Furthermore, the human brain material contains readily detectable N-terminally truncated amyloid-b species such as amyloid-b 4–40 and amyloid- b 4–42 , whereas CSF contains primarily full length amyloid-b and C-terminally truncated species such as amyloid-b 1–17 and amyloid- b 1–38 . The truncation analyses were based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. An important future direction will be to include higher resolution assessments of the material using techniques such as liquid chromatography with tandem mass spectrometry (LC-MS/MS) to build confidence in the identifications of amyloid-b species given the complexity of the material. Additional mass spectrometry approaches to identify other post-translational modifications and co-associated proteins will also be of great interest. Furthermore, while the observations of relative differences are likely to be solid and are based on well- designed controls, quantitation of amyloid-b at the attomole level is certainly challenging, particularly given its propensity for non- linear behaviour. Future refinements will be needed in order to perform quantitatively precise studies at this scale. The results presented by Fritschi et al. are perhaps the most dramatic example of an emerging theme; the vast functional di- versity within the ‘amyloid-b-ome’. Other examples include the following: Noguchi et al. (2009) reported that large soluble amyl- oid-b-containing assemblies termed amylospheroids, extracted from human Alzheimer’s disease brain lysates, caused apoptosis of rat septal neurons in culture at concentrations 40-fold lower than required for synthetic amyloid-b aggregates of similar size and immunoreactivity profile. Jin et al. (2011) revealed that small ( 8 kDa) amyloid-b immunoreactive species derived from human Alzheimer’s disease brain caused cytoskeletal disruption in cultured rat hippocampal neurons at concentrations 1000-fold lower than required for synthetic amyloid-b dimers. Moreover, Langer et al. (2011) found that a sub-fraction accounting for 5 0.05% of total brain amyloid-b from transgenic mouse brain was responsible for 30% of amyloid-b aggregate seeding activity in vivo (Langer et al., 2011). An important avenue for future investigations will be to deter- mine the specific relationship between these N-terminal modifica- tions of amyloid-b and induction of amyloid-b aggregation in vivo. The specific forms of amyloid-b detected by mass spec- trometry may be just the tip of the iceberg; many other post- translationally modified forms of amyloid-b have been described recently (Bayer and Wirths, 2014). Furthermore, the ultimate bio- logical activity is likely to be governed most directly by the 3D structures of amyloid-b assemblies, which may require advanced methods of assessment such as pulsed hydrogen–deuterium exchange and fast photochemical oxidation mass spectrometry for characterization (Gau et al., 2013; Zhang et al., 2013). Importantly, the relationship between aggregate seeding activity and synaptic toxicity, tau-related pathophysiological processes, and inflammatory responses remains to be established; it is pos- sible in principle that these events are dissociable with different structural determinants. However, oligomeric forms of amyloid-b derived from human Alzheimer’s disease brain seem to be the 2874 | Brain 2014: 137; 2872–2878 Scientific Commentaries by guest on November 2, 2014 Downloaded from

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Page 1: The remarkable properties of amyloid-  derived from human Alzheimer's disease brain: swinging the streetlight

The remarkable properties of amyloid-b derivedfrom human Alzheimer’s disease brain: swingingthe streetlight

This scientific commentary refers to ‘Highly potent soluble

amyloid-b seeds in human Alzheimer brain but not cerebrospinal

fluid’ by Fritschi et al. (doi: 10.1093/brain/awu255).

Despite many years of intensive study, the specific amyloid-bspecies in the human brain responsible for the pathophysiological

processes underlying Alzheimer’s disease have yet to be identified.

In part, this may be because we have been ‘searching under the

streetlight’: examination of other sources of amyloid-b such as

synthetic preparations, material derived from the brains of trans-

genic animals, and amyloid-b recovered from human CSF after

lumbar puncture has been much more convenient than direct

evaluation of human brain-derived material. However, telltale

signs over the last several years have indicated that amyloid-bderived from human brain may have properties or constituents

that are qualitatively and quantitatively different from those of

amyloid-b from other sources. The paper by Fritschi et al.

(2014) from Mathias Jucker’s group in this issue of Brain is a

major contribution to this line of investigation. Notably, the

paper provides perhaps the most definitive evidence yet that

human brain-derived amyloid-b has fundamentally dissimilar prop-

erties to human CSF-derived material.

Specifically, Fritschi et al. demonstrate that 51 attomole (10�18

moles) of water-soluble amyloid-b from frozen human Alzheimer’s

disease brain tissue is sufficient to induce accelerated amyloid-bplaque deposition when injected into the brains of young APP

transgenic mice. In contrast, human CSF from patients with de-

mentia of the Alzheimer’s type containing over 100 000 times

greater total mass of amyloid-b failed to affect amyloid-deposition

when injected in the same manner. Neither intrinsic anti-aggrega-

tion activity in CSF, nor freeze-thaw effects could explain these

results; and CSF from transgenic mice behaved similarly, excluding

any effect of species. The same lab has previously reported that

synthetic amyloid-b at 100–1000-fold higher concentrations also

fails to induce plaque deposition (Meyer-Luehmann et al., 2006).

Furthermore, neither synthetic amyloid-b dimers nor protofibrils,

mixed synthetic amyloid-b plus astrocyte-derived apolipoprotein E

particles, nor cell-culture derived amyloid-b immunoreactive spe-

cies induce in vivo plaque deposition (Meyer-Luehmann et al.,

2006).

The explanation for these remarkable observations is not at all

clear. By way of tantalizing hints, Fritschi et al. reveal that the

amyloid-b immunoreactive particles are larger in the human

Alzheimer’s disease brain lysates than in the CSF, and their

in vitro particle seeding activity is higher. Furthermore, the

human brain material contains readily detectable N-terminally

truncated amyloid-b species such as amyloid-b4–40 and amyloid-

b4–42, whereas CSF contains primarily full length amyloid-b and

C-terminally truncated species such as amyloid-b1–17 and amyloid-

b1–38. The truncation analyses were based on matrix-assisted laser

desorption/ionization (MALDI) mass spectrometry. An important

future direction will be to include higher resolution assessments of

the material using techniques such as liquid chromatography with

tandem mass spectrometry (LC-MS/MS) to build confidence in the

identifications of amyloid-b species given the complexity of the

material. Additional mass spectrometry approaches to identify

other post-translational modifications and co-associated proteins

will also be of great interest. Furthermore, while the observations

of relative differences are likely to be solid and are based on well-

designed controls, quantitation of amyloid-b at the attomole level

is certainly challenging, particularly given its propensity for non-

linear behaviour. Future refinements will be needed in order to

perform quantitatively precise studies at this scale.

The results presented by Fritschi et al. are perhaps the most

dramatic example of an emerging theme; the vast functional di-

versity within the ‘amyloid-b-ome’. Other examples include the

following: Noguchi et al. (2009) reported that large soluble amyl-

oid-b-containing assemblies termed amylospheroids, extracted

from human Alzheimer’s disease brain lysates, caused apoptosis

of rat septal neurons in culture at concentrations �40-fold lower

than required for synthetic amyloid-b aggregates of similar size

and immunoreactivity profile. Jin et al. (2011) revealed that

small (� 8 kDa) amyloid-b immunoreactive species derived from

human Alzheimer’s disease brain caused cytoskeletal disruption

in cultured rat hippocampal neurons at concentrations 1000-fold

lower than required for synthetic amyloid-b dimers. Moreover,

Langer et al. (2011) found that a sub-fraction accounting for

50.05% of total brain amyloid-b from transgenic mouse brain

was responsible for 30% of amyloid-b aggregate seeding activity

in vivo (Langer et al., 2011).

An important avenue for future investigations will be to deter-

mine the specific relationship between these N-terminal modifica-

tions of amyloid-b and induction of amyloid-b aggregation

in vivo. The specific forms of amyloid-b detected by mass spec-

trometry may be just the tip of the iceberg; many other post-

translationally modified forms of amyloid-b have been described

recently (Bayer and Wirths, 2014). Furthermore, the ultimate bio-

logical activity is likely to be governed most directly by the 3D

structures of amyloid-b assemblies, which may require advanced

methods of assessment such as pulsed hydrogen–deuterium

exchange and fast photochemical oxidation mass spectrometry

for characterization (Gau et al., 2013; Zhang et al., 2013).

Importantly, the relationship between aggregate seeding activity

and synaptic toxicity, tau-related pathophysiological processes,

and inflammatory responses remains to be established; it is pos-

sible in principle that these events are dissociable with different

structural determinants. However, oligomeric forms of amyloid-bderived from human Alzheimer’s disease brain seem to be the

2874 | Brain 2014: 137; 2872–2878 Scientific Commentaries

by guest on Novem

ber 2, 2014D

ownloaded from

Page 2: The remarkable properties of amyloid-  derived from human Alzheimer's disease brain: swinging the streetlight

most potent triggers for neurotoxicity, and in reports analogous to

the paper by Fritschi et al., amyloid-b oligomers seems to be pre-

sent in human Alzheimer’s disease CSF at concentrations many

orders of magnitude lower than in brain tissue lysates, if at all

(Xia et al., 2009; Esparza et al., 2013; Savage et al., 2014).

In conclusion, with the efforts of Fritschi et al., as well as several

other groups in recent years, the streetlight is now beginning to

swing around to illuminate the human brain directly.

Hopefully this is where we will find the keys to developing ef-

fective therapeutics for Alzheimer’s disease.

David L. Brody and Michael L. Gross

Washington University, USA

Correspondence to: David L. Brody

E-mail: [email protected]

doi:10.1093/brain/awu261

ReferencesBayer TA, Wirths O. Focusing the amyloid cascade hypothesis on

N-truncated Abeta peptides as drug targets against Alzheimer’s dis-

ease. Acta Neuropathologica 2014; 127: 787–801.

Esparza TJ, Zhao H, Cirrito JR, Cairns NJ, Bateman RJ, Holtzman DM,et al. Amyloid-beta oligomerization in Alzheimer dementia versus high-

pathology controls. Ann Neurol 2013; 73: 104–119.

Fritschi S, Langer F, Kaeser S, Maia L, Portelius E, Pinotsi D, et al. Highly

soluble Ab seeds in human Alzheimer’s brain but not cerebrospinalfluid. Brain 2014; 137: 2909–15.

Gau BC, Chen J, Gross ML. Fast photochemical oxidation of proteins for

comparing solvent-accessibility changes accompanying protein folding:

data processing and application to barstar. Biochim Biophys Acta 2013;

1834: 1230–8.

Jin M, Shepardson N, Yang T, Chen G, Walsh D, Selkoe DJ. Soluble

amyloid beta-protein dimers isolated from Alzheimer cortex directly

induce Tau hyperphosphorylation and neuritic degeneration. Proc

Natl Acad Sci USA 2011; 108: 5819–24.

Langer F, Eisele YS, Fritschi SK, Staufenbiel M, Walker LC, Jucker M.

Soluble Abeta seeds are potent inducers of cerebral beta-amyloid de-

position. J Neurosci 2011; 31: 14488–95.

Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S,

Schaefer C, Kilger E, et al. Exogenous induction of cerebral beta-

amyloidogenesis is governed by agent and host. Science 2006; 313:

1781–4.

Noguchi A, Matsumura S, Dezawa M, Tada M, Yanazawa M, Ito A,

et al. Isolation and characterization of patient-derived, toxic, high

mass amyloid beta-protein (Abeta) assembly from Alzheimer disease

brains. J Biol Chem 2009; 284: 32895–905.

Savage MJ, Kalinina J, Wolfe A, Tugusheva K, Korn R, Cash-Mason T,

et al. A sensitive abeta oligomer assay discriminates

Alzheimer’s and aged control cerebrospinal fluid. J Neurosci 2014;

34: 2884–97.Xia W, Yang T, Shankar G, Smith IM, Shen Y, Walsh DM, et al.

A specific enzyme-linked immunosorbent assay for

measuring beta-amyloid protein oligomers in human plasma and

brain tissue of patients with Alzheimer disease. Arch Neurol 2009;

66: 190–9.Zhang Y, Rempel DL, Zhang J, Sharma AK, Mirica LM, Gross ML. Pulsed

hydrogen-deuterium exchange mass spectrometry probes conform-

ational changes in amyloid beta (Abeta) peptide aggregation. Proc

Natl Acad Sci USA 2013; 110: 14604–9.

A serum microRNA signature for amyotrophiclateral sclersosis reveals convergent RNAprocessing defects and identifiespresymptomatic mutation carriers

This scientific commentary refers to ‘Serum microRNAs in pa-

tients with genetic amyotrophic lateral sclerosis and pre-manifest

mutation carriers’ by Freischmidt et al. (doi: 10.1093/brain/

awu249).

Defective RNA processing has occupied centre stage in the patho-

genesis of amyotrophic lateral sclerosis (ALS) since the identifica-

tion of TARDBP (also known as TDP-43) inclusions in 95% of

cases and pathogenic mutations in RNA processing genes such

as TARDBP, FUS and MATR3 (Sreedharan et al., 2008; Vance

et al., 2009; Johnson et al., 2014). FUS and TARDBP are known

to regulate mRNA transcription, splicing, stability and transport

(Tollervey et al., 2011; Rogelj et al., 2012) but they are also

part of the large Drosha complex that regulates microRNA

(miRNA) biogenesis (Gregory et al., 2004). Dysregulation of

miRNA expression has been shown in many cancers and more

recently in Alzheimer’s disease and is predicted to play a mechan-

istic role and/or be an indirect biomarker of disease.

In this issue of Brain, Freischmidt et al. (2014) report that levels

of a specific subset of miRNAs are reduced in the serum of pa-

tients with familial and sporadic ALS, and that these reductions are

even detectable in presymptomatic carriers of pathogenic ALS mu-

tations (Freischmidt et al., 2014). Some caution is required as pa-

tient numbers are small and some samples required pooling for

analysis, but if these results can be replicated in larger cohorts

then this will become a landmark study. Robust serum miRNA

biomarkers would aid early diagnosis and therefore early treat-

ment, and identify at-risk asymptomatic mutation carriers with

Scientific Commentaries Brain 2014: 137; 2872–2878 | 2875

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