Irene Otte-Höller

Specific association of small heat shock proteins with the pathological hallmarks of Alzheimer's disease brains

The small heat shock protein family (sHsp) comprises molecular chaperones able to interact with incorrectly folded proteins. Alzheimers disease (AD) is characterized by pathological lesions such as senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of the incorrectly folded proteins amyloid‐β (Aβ) and tau respectively. The aim of this study was to investigate the association of the chaperones Hsp20, HspB2, αB‐crystallin and Hsp27 with the pathological lesions of AD brains. For this purpose, a panel of well‐characterized antibodies directed against these sHsps was used in immunohistochemistry and immunoblotting. We observed extracellular expression of Hsp20, Hsp27 and HspB2 in classic SPs, and Hsp20 expression in diffuse SPs. In addition, extracellular expression of HspB2 was observed in CAA. Both Hsp27 and αB‐crystallin were also observed in astrocytes associated with both SPs and CAA. Furthermore, none of the sHsps were observed in NFTs in AD brains. We conclude that specific sHsp species may be involved in the pathogenesis of either SPs or CAA in AD.

Cerebral Microvascular Amyloid β Protein Deposition Induces Vascular Degeneration and Neuroinflammation in Transgenic Mice Expressing Human Vasculotropic Mutant Amyloid β Precursor Protein

Cerebral vascular amyloid beta-protein (Abeta) deposition, also known as cerebral amyloid angiopathy, is a common pathological feature of Alzheimers disease. Additionally, several familial forms of cerebral amyloid angiopathy exist including the Dutch (E22Q) and Iowa (D23N) mutations of Abeta. Increasing evidence has associated cerebral microvascular amyloid deposition with neuroinflammation and dementia in these disorders. We recently established a transgenic mouse model (Tg-SwDI) that expresses human vasculotropic Dutch/Iowa mutant amyloid beta-protein precursor in brain. Tg-SwDI mice were shown to develop early-onset deposition of Abeta exhibiting high association with cerebral microvessels. Here we present quantitative temporal analysis showing robust and progressive accumulation of cerebral microvascular fibrillar Abeta accompanied by decreased cerebral vascular densities, the presence of apoptotic cerebral vascular cells, and cerebral vascular cell loss in Tg-SwDI mice. Abundant neuroinflammatory reactive astrocytes and activated microglia strongly associated with the cerebral microvascular fibrillar Abeta deposits. In addition, Tg-SwDI mouse brain exhibited elevated levels of the inflammatory cytokines interleukin-1beta and -6. Together, these studies identify the Tg-SwDI mouse as a unique model to investigate selective accumulation of cerebral microvascular amyloid and the associated neuroinflammation.

Agrin Is a Major Heparan Sulfate Proteoglycan Accumulating in Alzheimer's Disease Brain

Heparan sulfate proteoglycans (HSPGs) have been suggested to play an important role in the formation and persistence of senile plaques and neurofibrillary tangles in dementia of the Alzheimers type (DAT). We performed a comparative immunohistochemical analysis of the expression of the HSPGs agrin, perlecan, glypican-1, and syndecans 1-3 in the lesions of DAT brain neocortex and hippocampus. Using a panel of specific antibodies directed against the protein backbone of the various HSPG species and against the glycosaminoglycan (GAG) side-chains, we demonstrated the following. The basement membrane-associated HSPG, agrin, is widely expressed in senile plaques, neurofibrillary tangles and cerebral blood vessels, whereas the expression of the other basement membrane-associated HSPG, perlecan, is lacking in senile plaques and neurofibrillary tangles and is restricted to the cerebral vasculature. Glypican and three different syndecans, all cell membrane-associated HSPG species, are also expressed in senile plaques and neurofibrillary tangles, albeit at a lower frequency than agrin. Heparan sulfate GAG side chains are also associated with both senile plaques and neurofibrillary tangles. Our results suggest that glycosaminoglycan side chains of the HSPGs agrin, syndecan, and glypican, but not perlecan, may play an important role in the formation of both senile plaques and neurofibrillary tangles. In addition, we speculate that agrin, because it contains nine protease-inhibiting domains, may protect the protein aggregates in senile plaques and neurofibrillary tangles against extracellular proteolytic degradation, leading to the persistence of these deposits.

Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity.

Alzheimer’s disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-β precursor protein (APP), the amyloid-β peptide (Aβ). CAA is also the major pathological lesion in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), caused by a mutation in the gene coding for the Aβ peptide. Several members of the small heat shock protein (sHsp) family, such as αB-crystallin, Hsp27, Hsp20 and HspB2, are associated with the pathological lesions of AD, and the direct interaction between sHsps and Aβ has been demonstrated in vitro. HspB8, also named Hsp22 of H11, is a recently discovered member of the sHsp family, which has chaperone activity and is observed in neuronal tissue. Furthermore, HspB8 affects protein aggregation, which has been shown by its ability to prevent formation of mutant huntingtin aggregates. The aim of this study was to investigate whether HspB8 is associated with the pathological lesions of AD and HCHWA-D and whether there are effects of HspB8 on Aβ aggregation and Aβ-mediated cytotoxicity. We observed the expression of HspB8 in classic SPs in AD brains. In addition, HspB8 was found in CAA in HCHWA-D brains, but not in AD brains. Direct interaction of HspB8 with Aβ1–42, Aβ1–40 and Aβ1–40 with the Dutch mutation was demonstrated by surface plasmon resonance. Furthermore, co-incubation of HspB8 with D-Aβ1–40 resulted in the complete inhibition of D-Aβ1–40-mediated death of cerebrovascular cells, likely mediated by a reduction in both the β-sheet formation of D-Aβ1–40 and its accumulation at the cell surface. In contrast, however, with Aβ1–42, HspB8 neither affected β-sheet formation nor Aβ-mediated cell death. We conclude that HspB8 might play an important role in regulating Aβ aggregation and, therefore, the development of classic SPs in AD and CAA in HCHWA-D.

Heparan sulfate proteoglycan expression in cerebrovascular amyloid β deposits in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis (Dutch) brains

Abstract.Cerebrovascular deposition of amyloid β protein (Aβ) is a characteristic lesion of Alzheimers disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). Besides Aβ, several other proteins and proteoglycans accumulate in cerebral amyloid angiopathy (CAA). We have now analyzed the expression of the heparan sulfate proteoglycan (HSPG) subtypes agrin, perlecan, glypican-1, syndecans 1–3 and HS glycosaminoglycan (GAG) side chains in CAA in brains of patients with AD and HCHWA-D. Hereto, specific well-characterized antibodies directed against the core protein of these HSPGs and against the GAG side chains were used for immunostaining. Glypican-1 was abundantly expressed in CAA both in AD and HCHWA-D brains, whereas perlecan and syndecans-1 and -3 were absent in both. Colocalization of agrin with vascular Aβ was clearly observed in CAA in HCHWA-D brains, but only in a minority of the AD cases. Conversely, syndecan-2 was frequently associated with vascular Aβ in AD, but did not colocalize with vascular Aβ deposits in HCHWA-D. The three different syndecans, agrin, glypican-1 and HS GAG, but not perlecan, were associated with the majority of senile plaques (SPs) in all brains. Our results suggest a role for agrin in the formation of SPs and of CAA in HCHWA-D, but not in the pathogenesis of CAA in AD. Both syndecan-2 and glypican, but not perlecan, may be involved in the formation of CAA. We conclude that specific HSPG species may be involved in the pathogenesis of CAA in both AD and HCHWA-D, and that the pathogenesis of CAA and SPs may differ with regard to the involvement of HSPG species.

Distribution of A beta-associated proteins in cerebrovascular amyloid of Alzheimer's disease.

Abstract Senile plaques and cerebrovascular amyloidosis (CA) are two of the major neuropathological lesions in brains of patients with dementia of the Alzheimer type. We studied the expression of a number of amyloid β (Aβ)-associated proteins in CA, which have previously been identified in senile plaques and which were suggested to play an important role in the pathogenesis of these lesions. Our findings show that involvement of inflammatory components in CA is restricted to activation of the complement system, resulting in deposition of the complement factors C1q, C3c, C4d and the membrane attack complex C5b-9 as well as of the complement inhibitor clusterin. Furthermore, we observed expression of apolipoprotein E, amyloid P component and heparan sulfate proteoglycans in CA, whereas expression of lactoferrin was almost absent. Other inflammatory proteins, known to be present in senile plaques, such as α1-antichymotrypsin, α2-macroglobulin and intercellular adhesion molecule-1, were absent or detectable only in small amounts. These data suggest that an incomplete inflammatory response occurs in CA as compared to senile plaques. This was confirmed by the finding that the number of cells of the monocyte/macrophage lineage around CA was not increased compared to unaffected vessels. Based on their expression patterns, complement factors, apolipoprotein E and heparan sulfate proteoglycans may be produced early in the process of CA formation and may play an important role in the formation of Aβ fibrils in CA. The absence of a number of Aβ-associated proteins in CA in comparison to senile plaques is in support of a different pathogenesis for these two lesions.

Accumulation of heparan sulfate proteoglycans in cerebellar senile plaques

Alzheimers disease (AD) brains are characterized by the presence of senile plaques (SPs), which primarily consist of amyloid beta protein (Abeta). Besides Abeta, several other proteins with the ability to modulate amyloid fibril formation accumulate in SPs, e.g. heparan sulfate proteoglycans (HSPGs). Cerebellar SPs are predominantly of the diffuse type, whereas fibrillar SPs are rarely observed. Furthermore, because of the spatial separation of non-fibrillar and fibrillar SPs in the cerebellum, this brain region provides a model for the study of the association of Abeta-associated factors with various stages of SP formation. In the present study, we performed an immunohistochemical analysis to investigate the expression of the HSPG species agrin, perlecan, glypican-1 and the syndecans 1-3 as well as glycosaminoglycan side-chains in cerebellar SPs. We demonstrated that agrin and glypican-1 were expressed in both non-fibrillar and fibrillar cerebellar SPs, whereas the syndecans were only associated with fibrillar cerebellar SPs. Perlecan expression was absent in all cerebellar SPs. Since fibrillar and non-fibrillar SPs may develop independently in the cerebellum, it is likely that agrin, glypican-1 as well as heparan sulfate glycosaminoglycans may contribute to the formation of both cerebellar plaque types, whereas syndecan only seems to play a role in the generation of cerebellar fibrillar plaques.

Amyloid-beta-induced degeneration of human brain pericytes is dependent on the apolipoprotein E genotype.

Abstract: Amyloid‐β (Aβ) deposition in cerebral vessels (cerebral amyloid angiopathy, CAA) is accompanied by degeneration of vascular cells, including pericytes and smooth muscle cells. Previous studies indicated that specific Aβ protein isoforms are toxic for cultured human brain pericytes and smooth muscle cells. In particular, Aβ1–40 carrying the E22Q mutation, as in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA‐D), is toxic. We investigated the effects of the Aβ‐binding protein apolipoprotein E (ApoE) on the toxicity of Aβ for cultured human brain pericytes. We compared the toxicity of HCHWA‐D Aβ1–40 for pericyte cultures with different ApoE genotypes, studied the accumulation of Aβ and ApoE in these different cell cultures, and investigated the effects of exogenous ApoE. Pericyte cultures with an ApoE ɛ2/ɛ3 genotype were more resistant to HCHWA‐D Aβ1–40 treatment than cultures with a ɛ3/ɛ3 or ɛ3/ɛ4 genotype. Cell death was highest in cultures homozygous for ApoE ɛ4. The extent to which both Aβ and ApoE accumulated at the cell surface was parallel to the degree of toxicity. The addition of purified ApoE resulted in a decrease in cell death. These data suggest that ApoE4 may direct Aβ more efficiently than other ApoE isoforms into a pathological interaction with the HBP cell surface. The results of this study are in line with the observations that inheritance of the ApoE ɛ4 allele increases the risk of developing Alzheimers disease, and that the ApoE ɛ2 allele has a relatively protective effect.

Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the A-betha-containing lesions in brains of patients with dementia of the Alzheimer type

Abstract Inflammatory processes have been implicated in the formation of senile plaques in the cerebral cortex of patients with dementia of the Alzheimer type (DAT), since several inflammation-induced proteins are present within these plaques. The relation between inflammatory components and other amyloid β protein (Aβ)-containing lesions of the DAT brain [cerebrovascular amyloidosis (CA) and cerebellar senile plaques] is unclear. We studied the distribution of the inflammation-inducible protein intercellular adhesion molecule-1 (ICAM-1) in CA and in senile plaques of the cerebellum, using an immunohistochemical approach. We observed striking differences in ICAM-1 reactivity between the different types of Aβ-containing lesions. ICAM-1 was only expressed in classic senile plaques in the granular and Purkinje cell layer of the cerebellum, and not in diffuse senile plaques of the molecular layer. Also, ICAM-1 was not associated with CA; only when the vascular amyloid extended into the neuropil (dyshoric angiopathy) was perivascular ICAM-1 reactivity observed. This is in contrast to the putative primary involvement of inflammation in the formation of cerebrocortical classic and diffuse senile plaques. Our findings indicate that ICAM-1 expression, which may be an indicator of an inflammatory reaction, is induced in the neuropil depending on the specific site of Aβ production.

Apolipoprotein E genotype regulates amyloid-beta cytotoxicity.

The ϵ4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimers disease (AD), whereas the ϵ2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with degeneration of smooth muscle cells and pericytes. Previously, we described that synthetic amyloid-β1-40 peptide (Aβ1-40) with the 22Glu→ Gln “Dutch” mutation caused pericyte death in vitro by a mechanism that involves Aβ fibril-like assembly at the cell surface. It is known that ApoE binds to Aβ and may modify its biological activities. In the present study, we evaluated the effect of ApoE on Aβ-mediated toxicity of cerebrovascular cells. We observed that cultured cells with an ϵ4/ϵ4 genotype were more vulnerable to Aβ than cultures with an ϵ3/ϵ3 or ϵ3/ϵ4 genotype. The one cell culture with the ϵ2/ϵ3 genotype was relatively resistant to Aβ compared with other cultures. Furthermore, we observed a dose-dependent protective effect of native ApoE against Aβ-mediated toxicity of cerebrovascular cells and, in addition, ApoE ϵ2/ϵ3 cells secreted more ApoE protein compared with cells with other ApoE genotypes, in particular, compared with ϵ4/ϵ4 cells. Thus, the disparity between ApoE genotype and Aβ-mediated toxicity might be related to differences in the cellular capacity to secrete ApoE. The present data suggest that one mechanism by which ApoE may alter the risk for AD is a genotype-dependent regulation of Aβ cytotoxicity, possibly via variations in its secretion levels, whereby extracellular ApoE may bind to Aβ and thereby modify Aβ-mediated cell death.