The multiple mechanisms of amyloid deposition

Abstract

Amyloid deposition is one of the central neuropathological abnormalities in Alzheimer disease (AD) but it also takes places in many neurodegenerative diseases such as prionic disorders, Huntington's disease (HD) and others. Up to very recently amyloid formation was considered a very slow process of deposition of an abnormal protein due to genetic abnormalities or post-translational modification of the deposited protein. Recent data suggest that the process of amyloidogenesis may be much more rapid in many cases and due to multiple mechanisms.

We have found a mouse model of progressive neurodegeneration that resemble motor, behavioral and pathological hallmarks of parkinsonism and tauopathies, but surprisingly, also present amyloid deposits in brain and peripheral organs. Here we review some of these recent works which may provide new insight into the process of formation of amyloid and, perhaps, new ideas for its treatment.

Introduction

The amyloidosis, or □-fibrilloses, are a group of diseases which share a common pathogenic mechanism: the deposition of one or more of a number of proteins which, as described by Virchow¹ more than one and a half centuries ago, stain with iodine compounds with similar characteristics to starch. The common ultra structure of amyloid proteins is made of some unbranched, rigid fibrils, 7.5 to 10 nm wide and of variable length² which arrange themselves in anti-parallel sheets with □ structure.³ The amyloid pathology could be present in many diseases but it could also appear in senile asymptomatic subjects who may show primarily the typical “senile amyloidotic triad”, brain, heart and pancreas.⁴ Amyloidosis could also occur in domestic animals.⁵

The kinetics of the amyloid deposition process in vivo demonstrates the temporal relations between amyloid deposits, microglial activation and neuritic changes.^6,7 According to these data amyloid deposition, under certain circumstances, could take place in a short period of time. These results are surprising because it has been generally accepted, based on in vitro studies of protein aggregation, that amyloid-□ aggregation is time dependent and follows a relatively slow nucleation-dependent polymerization process.⁸ But the most recent data could help to explain the formation of amyloid events, which takes place after short-lived amyloidogenic insults. For instance, the risk of Alzheimer's disease (AD) has been reported to increase with stroke and anesthesia.^9–13

Clinical Classification of the Amyloidosis

The amyloidosis are classified according to a recent international nomenclature according to which the first letter symbolizes the kind of amyloid deposits and the following letters, the type of proteins involved.¹⁴ Recent reviews of the topic include amyloidosis produced by more than 25 human fibrillary proteins plus eight or more additional in aminals.¹⁵ For a fibrillary protein to be considered amyloidogenic it should produce extracellular deposits with affinity for the red Congo dye and a green birefringence. According to the actual terminology, AL amyloidosis indicates amyloid produced by light chains of immunoglobulins. From the clinical point of view, a classification that takes into consideration the organs or systems involved and the pattern of inheritance is important, as is proposed in Table 1.

Table 1.

Clinical classification of the amyloidosis

Tissue involvement	Pattern of inheritance	Pathogenic mechanism
Systemic amyloidosis	Hereditary, autosomal dominant	Mutations of the transtiretin, A□ chain of fibrinogen, apolipoproteins I and II, and gelsolin genes
	Primary, sporadic	Accumulation of the light chains of the immuno-globulins in multiple myeloma.
	Secondary, sporadic	Accumulation of immunoglobulins which take place while aging and in chronic infectious and inflammatory processes.
Oligovisceral amyloidosis	Hereditary and sporadic, at times with the same protein.	Accumulation of different proteins which deposit selectively or preferently in different organs, according to their genic expression or their metabolism.

Cerebral Amyloidosis Associated to Neurodegenerative Diseases

The amyloidosis which cause neurodegenerative disorders are almost always related to intracerebral production of the pathogenic protein since most proteins, like immunoglobulins, do not cross the blood brain barrier. One exception may be the case of systemic amyloidosis related to transtiretin mutations, which produces peripheral neuropathy and cerebral changes in the white matter in some cases.¹⁶ A list of the amyloidosis associated to neurodegeneration is presented in Table 2.

Table 2.

Cerebral amyloidoses associated to neurodegenerative diseases

Disease	Pathogenic protein	Pathogenic mechanism
Alzheimer's type dementia	□-amyloid peptide	Excessive production or accumulation of the peptide due to mutations of the precursor proteins, or the processing proteins.
Spongiform encephalopathies	Prion protein	Mutations of the peptide, modeling in □ planar structures according to the infectious protein
Familial amyloid angiopathy	□-amyloid peptide	Mutations of the cystatine C, an inhibitor of the proteasas which metabolizes the □-amyloid peptide
British and Danish familial amyloid dementias	Peptides derived from BriPP (chromosome 13)	Peptide of 34 amino acids in the C terminal region of the protein ABriPP, which deposits in the blood vessels and brain parenchyma of the patients with these mutations
Tauopathies	Protein tau (fibrillary)	Tau protein changes its conformation and adopts a fibrilar structure with alterations of b-amyloid peptide, □-synuclein and parkin
Synucleinopathies	Normal and mutanst □-synuclein	Mutant □-synuclein (point mutations A53T, A30P or E46K; duplication or triplication of the gene). Post-translationally altered □-synuclein mostly by nitration or phosphorylation
Huntington's disease	Mutant huntingtin	Increased resistance to degradation

The model of cerebral amyloidosis is AD, which is in fact, a complex of diseases related to multiple mechanisms, characterized by lack of memory, aphasia, apraxia, agnosia, associated with a loss of neurons in the cerebral cortex, mostly in multimodal association areas of the parietal and temporal cortex as well as in the hippocampus and the entorhinal cortex. In these regions, there is an extracellular deposition of □-amyloid peptide, the senile plaques, as well as intracellular deposition of neurofibrillary tangles. The protein involved in neuro-fibrillary tangles is mostly tau protein, and the cognitive deficits in AD are better correlated with neurofibrillary pathology than with amyloid plaques.^17,18

The link between tau pathology and □-amyloid (A□) accumulation has been largely investigated because it is the pathological characteristic and probably one of the keys to understanding the pathogenesis of Alzheimer's disease, the most prevalent neurodegenerative disease world wide. There are still controversies about the bi-directional relation between both pathologies. Several works in transgenic animals supported the upstream role of □-amyloid. Gotz et al.¹⁹ and have shown that injection of A□ into the brain of tau transgenic mice exacerbates tau pathology, whereas Lewis et al.²⁰ have found enhanced tau pathology in mice expressing amyloid precursor protein (APP) and tau transgenes compared with single tau transgenic mice. The clearance of A□, using A□-specific antibodies reduced the tau burden,²¹ and genetically augmenting tau levels does not modulate the onset or progression of A□ pathology in a model of transgenic mice.²²

A□ and tau form a soluble complex that may promote self-aggregation of both.²³ Recently, it has been shown that reducing endogenous tau ameliorates A□-induced behavioral deficits in a mouse model of AD, which suggests a mutual interrelation between these two proteins.²⁴

Mechanisms of Amyloid Production in Neurodegenerative Disorders

The steady state levels of □-amyloid peptide in the brain represents a balance between its biosynthesis from the amyloid precursor protein (APP), its oligomerization into neurotoxic and stable species and its degradation. This peptide is a fragment of variable length, usually from 39 to 43 amino acids, located in the amino terminal, extra-membranous region of the APP. □-amyloid peptide is produced by enzymatic processing of APP mediated by the □-and □-secretases. □-Secretase is an enzymatic complex in which the presenilins, nicastrin, APH-1 (anterior pharinx defective-1) and PEN-2 (presenilin enhancer 2 gene) are the necessary and sufficient components for its enzymatic activity. Still today, there is debate about the exact location of □-amyloid production, the lysosomes or the plasma membrane. Mutations of the presenilins, which increase enzyme activity and increase □-amyloid production, as well as APP mutations, are known to produce AD (Table 3).

Table 3.

Mechanisms of amyloid production in neurodegenerative disorders

Mechanisms of disease	Mechanism of production of excessive or abnormal protein	Disease
Excessive production of normal protein	Trisomy of chromosome 21. Triplication APP gen Duplication or triplication of □-synuclein Excessive synthesis of immunoglobulins	Down's syndrome Familial Parkinson's disease Multiple myeloma
Pathogenic mutations of the abnormal protein	Mutations of APP, presenilins 1 and 2 Mutations of PrP gene Mutations of □-synuclein	Hereditary Alzheimer's disease Hereditary prionic disease Familial Parkinson's disease
Conformation of normal protein according to the model of the abnormal form	Ingestion or injection of abnormal PrP	Bovine spongiform encephalopathy (mad cow disease) Sporadic and iatrogenic Creutzfeldt Jacob disease
Abnormalities of the protein processing system (chaperones, UPS, etc.,) or polymorphisms of interacting proteins	Polymorphisms of parkin Polymorphisms of ApoE (ApoE 4 allele)	Tauopathies Alzheimer's disease
Post-translational changes of genetically normal proteins (hyperphosphorylation, nitration, oxidation, etc.)	Post-translational changes of □-synuclein	Sporadic Parkinson's disease

The variety of amyloid-degrading enzymes include: neprilysin (NEP) and its homologue endothelin-converting enzyme (ECE), insulin degrading enzyme (IDE), angiotensin-converting enzyme (ACE), matrix metalloproteinase-9 (MMP-9), the endo-lisosomal cathepsins, and the serine proteinase plasmin. These various amyloid-degrading enzymes have distinct subcellular localizations, and differential responses to aging, oxidative stress and pharmacological agents.Their upregulation may provide a novel and viable therapeutic strategy for prevention and treatment of Alzheimer's disease.²⁵

Human A□ (1–40, 42) monomers are also eliminated from the brain across the blood brain barrier (BBB). The BBB efflux clearance is reduced without downregulation and/or dysfunction of A□ efflux-transport system at the BBB. Thus, compounds that block the aggregation of soluble A□ monomers or the interaction between soluble A□ and its binding molecules could also be clinically useful.^26,27

Deposition of amyloid, a critical neuropathological finding in AD, could be related to several mechanisms and not only to abnormal sequence or excessive production of the involved protein. It may also appear in conditions when the chaperoning or proteosomal processing of denatured proteins do not work efficiently and in conditions of sustained oxidative stress.

Amyloidosis and Proteosome

The ubiquitin-proteasome pathway (UPS) is involved in a vast array of cellular processes, including protein trafficking, antigen presentation and protein degradation of short-lived proteins. The proteasome is a large protease complex responsible for intracellular degradation of misfolded, oxidized or aggregated proteins.²⁸ Aging impairs proteasome function at several levels, with effects on proteasome expression, activity and response to oxidative stress.^29,30

There is growing evidence suggesting that the ubiquitinproteasome pathway is linked to the neuro-pathogenesis of AD. The accumulation of ubiquitin and ubiquitinated proteins and the presence of the aberrant form of ubiquitin (UBB + 1) in AD brain suggest the involvement of the ubiquitin-proteasome pathway in AD.³¹ Several studies have shown UPS dependent degradation of proteins related to AD pathology including: tau,^32,33 the C-terminus fragment of APP,^34–36 BACE1,³⁷ PEN2,^38,39 PS1 and PS2,^40,41 APH-1,⁴² and nicastrin.⁴³ This degradation is mediated by different E3 ubiquitin ligases.

Parkin and Amyloidosis

Parkin was discovered as a protein with ubiquitin ligase E3 function, whose deletions or mutations produced autosomal recessive Parkinson's disease (PD) in humans.^44–48 Parkin mutations and parkin polymorphism increase the risk of deposition of tau in progressive supranuclear palsy (PSP).^49,50 Furthermore, in mice overexpressing human mutated tau (Tau^VLW), which in normal conditions do not form amyloid deposits, the suppression of parkin (PK^-/-) aggravates the behavioral deficits and produces severe cerebral and generalized amyloidosis.⁵¹

Mutations of tau and parkin proteins produce neuro-fibrillary abnormalities without deposition of amyloid. PK^-/-/Tau^VLW mice have abnormal behavior and dopamine neurotransmission with a severe drop-out of dopamine neurons in the ventral midbrain, of up to 70%, at 12 months and abundant astrogliosis and microgliosis.^51,52 The loss of DA neurons in the substantia nigra after tau overexpression in a rat model is restored by parkin overexpression⁵³ and the lack of parkin leads to tau accumulation with aging in mice.⁵⁴ PK^-/-/Tau^VLW mice present more phosphorylated tau positive neuritic plaques and neuro-fibrillary tangles than Tau^VLW mice, but, interestingly they also present □-amyloid plaques in the hippocampus (Fig. 1). We have found deposition of amyloid in brain of PK^-/-/Tau^VLW mice but no differences in the levels of APP or soluble A□ peptide, suggesting that our findings are not related to overproduction of the pathological peptide or its precursor protein but to increased deposition.

Figure 1.

Tau and □-amyloid pathology in the hippocampus of 12-month-old Tau^VLW and PK^-/-/Tau^VLW mice. Representative microphotographs of the Tau^VLW (A) and PK^-/-/Tau^VLW (B) hippocampus immunostained with antibody anti-tau phosphorylated in Ser 212 (AT-8). (C) Representative neuritic plaque. (D) The number of senile plaques, immunostained with AT-8, was statistically higher in PK^-/-/Tau^VLW mice. Representative microphotographs of hippocampus of 12-month-old TauVLW (E) and PK^-/-/Tau^VLW (F) Gallyas stained. Higher n^o of fibers and neuro-fibrillary tangles could be observed in PK^-/-/Tau^VLW hilus. Representative microphotographs of the hippocampus of Tau^VLW (G), PK^-/-/Tau^VLW (H and I) mice immunostained with antibody anti 1–40 and 1–42 □-amyloid peptides. (I) Magnification of square in H. Values are the mean ± SEM (n = three mice, three slice/mice, twice). (J) The number of amyloid plaques, immunostained with antibody anti 1–40 and 1–42 □-amyloid peptides, was statistically higher in 14-month-old PK^-/-/Tau^VLW mice. Statistical analysis was performed by t test. ***p < 0.001 PK^-/-Tau^VLW vs Tau^VLW mice. Scale = 200 µm for (A, B, G and H); and 50 µm for (C, E, F and I).

To our knowledge, PK^-/-/Tau^VLW mice are the first model that has developed A□ pathology without amyloid precursor protein or directly related proteins manipulation. Surprisingly, the amyloid deposits are not restricted to the brain. PK^-/-/Tau^VLW mice have organomegaly of the liver, spleen and kidneys. The electron microscopy of the liver confirmed the presence of extracellular fibrillary protein deposits with amyloid characteristics (Fig. 2). These peripheral effects of PK^-/- deletion can be explained, because parkin mRNA is expressed in liver, kidney and testis (Drosophila PK^-/- flies are sterile) at higher levels than in the brain.^55,56 Accordingly, parkin mice knock-out muscle cells are also more sensitive to the toxic effects of intracellular A□, parkin overexpression in skeletal muscle cultures provides substantial protection against A□ toxicity, and accumulation of parkin protein are present in skeletal muscle biopsies taken from patients with inclusion body myositis.^57,58

Figure 2.

Systemic hepatomegaly and amyloid fiber deposition in PK^-/-/Tau^VLW mice. Weight (A) and hematoxilin eosin stain histology of liver from nine-month-old Tau^VLW (C), and PK^-/-/Tau^VLW (B and D) mice showing the hypertrophy and the disorganization of liver structure in PK^-/-/Tau^VLW mice. Values are the mean ± SEM (n = six animals per experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p < 0.05, transgenic vs WT mice; ⁺p < 0.05, PK^-/-Tau^VLW vs PK^-/-mice, ^p < 0.05, PK^-/-Tau^VLW vs Tau^VLW mice. Scale bar = 50 µm. (E) In the electronic microscopy magnification we can observe membranous structures around mitochondrion, like pre-autophagosomes in Tau^VLW livers. (F) In PK^-/-/Tau^VLW livers, there are fibrillar deposits compatible with amyloids in the Disse space.

There is also accumulation of mouse tau in PK^-/-/Tau^VLW hepatocytes (Fig. 3). Tau is physiologically expressed in mouse livers.^59,60 Mouse tau levels are four times higher in PK^-/-/Tau^VLW livers than in the other groups, and tau forms cytoplasm inclusions, only detectable in PK^-/-/Tau^VLW hepatocytes. However, mRNA protein inclusions promoting its degradation by autophagy.^75,76 We have observed p62 accumulation in PK^-/-/Tau^VLW livers (Fig. 3). It could be a cellular response to tau accumulation trying to compensate the reduced levels of parkin, CHIP and HSP70 by increasing autophagy.

Figure 3.

Expression of tau and proteins involved in tau degradation in livers and brains of WT, PK^-/-, Tau^VLW and PK^-/-/Tau^VLW nine-month-old mice. Representative microphotographs of anti-tau immunohistochemistry with a polyclonal antibody in WT (A), PK^-/- (B), Tau^VLW (C) and PK^-/-/Tau^VLW (D) livers. Scale = 15 µm. (E) Representative western blot of tau in livers and brains from WT, PK^-/-, Tau^VLW and PK^-/-/Tau^VLW mice revealed with an anti-tau polyclonal antibody and the densitometric histograms of mouse tau. Observe the different scale in liver and brain histograms. Representative western blot and densitometric histograms of CHIP (F), HSP70 (G) and p62 (H) in livers. CHIP and HSP70 expressions are lower, while p62 levels are higher in PK^-/-/Tau^VLW livers. (I) GSH levels in WT, PK^-/-, Tau^VLW and PK^-/-/Tau^VLW mice livers. Values are the mean ± SEM (n = 6 animals per experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. **p < 0.01, ***p < 0.001 transgenic vs. WT mice; ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 PK^-/-Tau^VLW vs PK^-/- mice, ^^^p < 0.001 PK^-/-Tau^VLW vs. Tau^VLW mice. The two-way ANOVA showed interaction between parkin deletion and Tau^VLW overexpression effects in the tau quantity in livers (**p < 0.01).

Parkin, as well as p62, mediate K63-linked polyubiquitination, a signal for targeting misfolded proteins to the aggresome-autophagy pathway.⁷⁷ The mouse models in which autophagy is inhibited, show, in addition to neurodegeneration, a hepatomegaly similar to that which we observe in PK^-/-/Tau^VLW mice.⁷⁸ We have shown⁵¹ that the increased lysosomal activity⁷⁹ and the elevated number of autophagosomes in the Tau^VLW livers turn to amyloid deposition in the absence of parkin (Fig. 2).

Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.⁸⁰ PK^-/- neurons are more susceptible to mitochondrial toxins⁸¹ and to oxidative stress and they have decreased levels of glutathione during aging.⁵⁴ PK^-/-/Tau^VLW mice also have reduced glutathione in the brain and in the liver.^51,52 The elevated levels of free radicals in absence of parkin could contribute to the amyloidosis. Other mechanisms though parkin could be involved in amyloid deposition, are the altered inflammatory response and elevated microglial levels observed in absence of parkin^82–84 which have been extensively implied in AD⁸⁵ and are also observed in the PK^-/-/Tau^VLW mice model.⁵¹

This model suggests that parkin could be a link between tau and amyloid deposition, the two most important pathogenic mechanisms in AD. It is also the first that demonstrates □-amyloid deposits caused by overexpression of tau and without modification of the amyloid precursor protein, presenilins or secretases. Since parkin suppression increases tau aggregation and amyloid deposition, why is it considered that patients with autosomal recessive parkinsonism due to parkin mutations is not characterized by dementia levels of tau or Tau^VLW do not change. The ubiquitination grade of tau leads to their degradation by proteasome or by lysosomes.³² The complex CHIP/HSP70,^61–66 and the Sequestosome/p62,^67–69 have been involved in tau ubiquitination and degradation through the proteasome. The lower expression of CHIP and HSP/70 in PK^-/-/Tau^VLW livers (Fig. 3) could promote tau accumulation in the absence of parkin. CHIP and HSPs also interact with A□ and influence A□ metabolism.^70,71 Recently, Oddo et al.⁷² have proposed a key role of CHIP as link of A□ and tau pathologies in a triple transgenic AD model. CHIP and HSP70 interact with parkin and modulate its E3 ubiquitin ligase and the proteosomal degradation of its substrates.⁷³

A decrease of proteosome activity causes tau accumulation.³² A reduction of Sequestosome/p62 expression also causes tau deposition.^67,74 Sequestosome/p62 is a common component of cytoplasmic inclusions in PD and AD and forms a patch around of Alzheimer type? One possible explanation is that the patients with autosomal recessive parkinsonism due to parkin mutations are “too” young for Alzheimer disease, by average 32 years of age, much younger that the age at onset of Alzheimer's disease even in cases related with mutations of APP and presenilins 1 and 2. In addition, although it is true that most clinical reports of patients with parkin mutations do not describe dementia as a characteristic clinical feature of this disease more recent studies suggest the opposite. Benbunan et al.⁸⁶ reported two brothers, aged 51 and 55, with deletions of exons 4–6 of parkin and cognitive decline. We have found a family with three siblings out of seven, with a single hemizygotic deletion 255A of parkin, with clear dementia in two of them in the eigth decade of life (JG Yebenes, personal observation). Even the pathological reports not always show, as it was considered initially, restricted lesions of the substantia nigra and brain stem nuclei in patients with parkin mutations. Mori et al.⁸⁷ found neurofibrillary tangles and argyrophillic astrocytes in the cerebral cortex. Van Warremburg et al.⁸⁸ also found tau pathology in the cortex.

We hope that our work could help towards explaining the basic pathogenic mechanisms, which trigger amyloidosis.

Abbreviations

A□

□-amyloid peptide

AD

alzheimer disease

APP

amyloid precursor protein

BBB

blood-brain barrier

CHIP

c-terminal Hsc interacting protein

PD

Parkinson disease

PK^-/-

parkin null mice

HSP

heat shock proteins

P62

sequestosome protein

PSP

progressive supranuclear palsy

Tau^VLW

mice overexpressing human mutated tau

TH

tyrosine hydroxylase

UPS

ubiquitin proteasome system

WT

wild-type