Heat Shock Proteins and Amateur Chaperones in Amyloid-Beta Accumulation and Clearance in Alzheimer 's Disease The pathologic lesions of Alzheimer 's disease -LRB- AD -RRB- are characterized by accumulation of protein aggregates consisting of intracellular or extracellular misfolded proteins . The amyloid-β -LRB- Aβ -RRB- protein accumulates extracellularly in senile plaques and cerebral amyloid angiopathy , whereas the hyperphosphorylated tau protein accumulates intracellularly as neurofibrillary tangles . `` Professional chaperones '' , such as the heat shock protein family , have a function in the prevention of protein misfolding and subsequent aggregation . `` Amateur '' chaperones , such as apolipoproteins and heparan sulfate proteoglycans , bind amyloidogenic proteins and may affect their aggregation process . Professional and amateur chaperones not only colocalize with the pathological lesions of AD , but may also be involved in conformational changes of Aβ , and in the clearance of Aβ from the brain via phagocytosis or active transport across the blood -- brain barrier . Thus , both professional and amateur chaperones may be involved in the aggregation , accumulation , persistence , and clearance of Aβ and tau and in other Aβ-associated reactions such as inflammation associated with AD lesions , and may , therefore , serve as potential targets for therapeutic intervention . Introduction Pathological lesions consisting of intra - and/or extracellular accumulations of misfolded proteins are characteristic for neurodegenerative diseases such as Alzheimer 's disease -LRB- AD -RRB- . AD is characterized by three distinct pathological lesions : senile plaques -LRB- SPs -RRB- , neurofibrillary tangles -LRB- NFTs -RRB- , and cerebrovascular amyloid angiopathy -LRB- CAA -RRB- -LSB- 1 -RSB- . Both SPs and CAA are formed by extracellular deposition of aggregated amyloid-beta protein -LRB- Aβ -RRB- , whereas NFTs consist of intracellular aggregates of hyperphosphorylated tau protein in the cytoplasm of neurons -LSB- 2 , 3 -RSB- . The Aβ protein is a 4-kDa proteolytic cleavage product -LSB- 2 -RSB- of the transmembrane amyloid-β precursor protein -LRB- APP -RRB- . The two major forms of Aβ in human brain are Aβ1-40 and Aβ1-42 , differing from each other only by two amino acids . Cerebral production of Aβ is balanced by clearance from the brain either via active transport across the blood -- brain barrier -LRB- BBB -RRB- or via uptake and degradation of Aβ by microglial cells and astrocytes -LSB- 4 -- 6 -RSB- . Active transport of Aβ is mediated by Aβ receptors that are capable of transporting Aβ , or Aβ in complex with other proteins , across the BBB -LSB- 7 -RSB- . In contrast to normal brain , the cerebral Aβ balance is disturbed in AD brains , resulting in accumulation and aggregation of Aβ . Aβ aggregation includes the formation of Aβ oligomers , protofibrils , and eventually , mature fibrils . Both Aβ oligomers and protofibrils are considered the most toxic forms of Aβ that initiate degeneration of neurons and of cells within the vasculature , such as smooth muscle cells and pericytes -LSB- 8 , 9 -RSB- . Aβ aggregates do not clear from the brain as efficiently as soluble Aβ , and thus , directly lead to increased levels of Aβ in the brain -LSB- 10 -RSB- . Furthermore , deposition of Aβ in SPs is accompanied by attraction and activation of both microglial cells and astrocytes -LSB- 11 -- 13 -RSB- . Activation of these cell types results in increased secretion of pro-inflammatory cytokines as part of a neuro-inflammatory reaction . Chaperones can be defined as proteins that : -LRB- 1 -RRB- have a role in the intracellular handling of misfolded proteins , -LRB- 2 -RRB- induce conformational changes of proteins , -LRB- 3 -RRB- act as transporter of proteins . `` Professional '' chaperones , such as the heat shock protein family -LRB- Hsp -RRB- , are defined as proteins that have a specific function in facilitating normal folding of proteins and intracellular handling of misfolded proteins . Members of the Hsp family recognize misfolded proteins and transport them to the proteasome for degradation . Therefore , this protein family acts as the first line of defense against toxicity induced by misfolded proteins such as Aβ and tau . In contrast to professional chaperones , `` amateur '' chaperones can be defined as proteins that bind to other proteins and induce conformational changes or , alternatively , serve as transporter proteins . Examples of putative amateur chaperones are apolipoprotein E -LRB- ApoE -RRB- , heparan sulfate proteoglycans -LRB- HSPGs -RRB- , and complement factors such as C1q . They have , in contrast to the professional chaperones , primarly an extracellular function . In this paper , we will review the role of both amateur and professional chaperones in the pathogenesis of AD . Aβ-Binding Proteins in Extracellular Interaction with Aβ Apolipoproteins The apolipoprotein family consists of proteins that conjugate with lipids to form different classes of lipoprotein particles . In human brain , several members of this protein family are expressed , such as apolipoprotein E -LRB- ApoE -RRB- , apolipoprotein J -LRB- ApoJ -RRB- , and apolipoprotein D -LRB- apoD -RRB- . ApoE is a major determinant of lipid transport and metabolism and is expressed in brain by astrocytes , microglia , pericytes , and smooth muscle cells -LSB- 14 -- 18 -RSB- . In human , three common isoforms are expressed : apoE2 , apoE3 , and apoE4 which are all products of alleles at a single gene locus -LSB- 19 , 20 -RSB- . The ɛ4 allele of ApoE is the major genetic risk factor for AD , whereas the ɛ2 allele appears to be protective against AD -LSB- 21 -- 24 -RSB- . As ApoE immunoreactivity was found in extracellular amyloid deposits in subjects with AD , it has been suggested that it affects amyloidogenesis -LSB- 25 , 26 -RSB- . In vitro studies provided evidence for a direct interaction of ApoE with Aβ and the formation of stable complexes -LSB- 27 , 28 -RSB- . Binding of ApoE to Aβ is , however , ApoE isoform-dependent -LRB- ɛ2 > ɛ3 >> ɛ4 -RRB- -LSB- 29 , 30 -RSB- and depends on the degree of lipidation -LSB- 29 -RSB- . Lipidation of ApoE also seems a major factor in its effect on Aβ-mediated cellular toxicity -LSB- 18 -RSB- . In addition , ApoE4 promotes the conversion of soluble Aβ into β-sheet-rich amyloid more than ApoE3 -LSB- 31 -- 33 -RSB- . In contrast to its effect on Aβ in vitro where a consistent accelerating effect of ApoE on Aβ aggregation is observed , the effect of ApoE on Aβ deposition in transgenic -LRB- Tg -RRB- mice studies is less equivocal . In early studies , both Aβ immunoreactivity and amyloid formation were reduced in ApoE knockout mice -LSB- 31 , 32 -RSB- . In addition , CAA and associated microhemorrhages were also suppressed in ApoE knockout mice -LSB- 34 -RSB- . This effect might be due to the absence of ApoE/Aβ complexes -LSB- 35 -RSB- . In contrast to the effects of murine ApoE in the early studies , both human ApoE3 and ApoE4 suppressed Aβ deposition in Tg mice -LSB- 36 -RSB- . In addition , when these mice aged , ApoE4 induced a tenfold higher deposition of fibrillar Aβ than ApoE3 -LSB- 37 -RSB- . Consistent with these latter results , human ApoE4 accelerated Aβ deposition in APPSwe Tg mice relative to human ApoE3 -LSB- 38 -RSB- . In addition , when human ApoE3 or ApoE4 were knocked in in Tg mice , Aβ deposition was reduced compared to mice carrying endogenous ApoE at 9 months , and at 15 months , substantial CAA was observed in mice with human ApoE4 , but not with human ApoE3 , and , in either case , parenchymal Aβ was sparse -LSB- 39 -RSB- . Thus isoform - and species-specific differences in ApoE direct the aggregation or clearance of Aβ . Furthermore , it is suggested that the presence of ApoE facilitates internalization and degradation of Aβ from brain parenchyma by astrocytes -LSB- 40 -RSB- and human ApoE may reduce Aβ deposition in mouse brain by facilitating Aβ transport across the BBB -LSB- 36 -RSB- . Although these Tg mice studies seem contradictional , ApoE clearly affects conformational changes of Aβ and functions as an Aβ-transporter protein . Besides colocalization of ApoE with Aβ in AD brains , ApoE is also found within neurons containing NFTs -LSB- 25 -RSB- where it is able to interact directly with tau protein -LSB- 41 -RSB- . Furthermore , ApoE has an isoform-dependent effect on tau phosphorylation , as ApoE3 binds to tau in vitro , whereas ApoE4 fails to bind tau -LSB- 42 -RSB- . In addition , an ApoE4-dependent increase in phosphorylated tau has been observed -LSB- 43 -- 45 -RSB- . Neuroinflammation in AD comprises both activation of microglial cells and astrocytes and activation of the complement system . Aβ deposits in brain are associated with activated microglia and astrocytes , but also with elevated levels of complement -LSB- 5 , 6 , 46 -RSB- . ApoE may have an anti-inflammatory effect by suppressing microglial and astrocytic activation -LSB- 47 -- 50 -RSB- . ApoE-deficient mice demonstrate increased levels of IL-6 and TNFα after LPS stimulation , suggesting a role of ApoE in inflammatory gene regulation -LSB- 51 -RSB- . In addition , ApoE isoform-dependent -LRB- ɛ2 < ɛ3 < ɛ4 -RRB- differences in nitric oxide -LRB- NO -RRB- levels have been observed in microglia cells -LSB- 52 -RSB- . Transgenic mice expressing the ApoE4 protein isoform show a greater NO production than mice expressing the ApoE3 protein isoform . These data indicate that ApoE4 has a less efficient anti-inflammatory affect , and thus , may accelerate the development of AD . Apolipoprotein J , also known as clusterin or SP-40 / 40 , is a highly conserved heterodimeric secreted glycoprotein expressed in brain by epithelial and neuronal cells -LSB- 53 -RSB- . ApoJ colocalizes with fibrillar Aβ deposits , and it is suggested that it prevents misfolding and aggregation of soluble Aβ -LSB- 54 -- 56 -RSB- . ApoD is a glycoprotein associated with high-density lipoproteins in human plasma and also has a high expression level in human brain -LSB- 57 -RSB- , but neither its physiological role nor its ligand has been identified . ApoD levels are increased in the hippocampus of AD patients and in ApoE-deficient mice -LSB- 58 , 59 -RSB- . In conclusion , ApoE and ApoJ can be regarded as amateur chaperones that regulate Aβ aggregation in vitro . By accelerating the Aβ aggregation process towards mature fibril formation , -LRB- human -RRB- ApoE prevents formation of toxic Aβ intermediates such as oligomers and protofibrils , and thus , may have a protective function towards development of AD . Moreover , ApoE protects against the development of AD by suppressing the inflammatory reactions associated with AD lesions . Besides its role in inducing conformational changes in Aβ , ApoE facilitates Aβ clearance from brain by serving as a transporter molecule of Aβ , which will be discussed in paragraph 4 . Heparan Sulfate Proteoglycans Proteoglycans are members of a large family of macromolecules with a wide variety of functions ranging from simple physical support to various effects on cell adhesion , motility , proliferation , differentiation , and even tissue morphogenesis . They are composed of linear sulfated polysaccharides -LRB- glycosaminoglycans , GAGs -RRB- , consisting of disaccharide units , covalently bound to a core protein . One of the members of this superfamily is the heparan sulfate proteoglycan -LRB- HSPG -RRB- family characterized by polymers of repeating disaccharides , N-acetylglucosamine and glucuronic acid , which can be subsequently modified by sulfatation -LSB- 60 , 61 -RSB- . HSPGs can be subdivided into a family of extracellular matrix proteins , including perlecan , agrin , and collagen XVIII , and a family of cell surface proteins , including syndecans and glypicans -LSB- 60 , 62 -RSB- . Ever since GAGs were demonstrated in amyloid deposits , the proteoglycans became of interest in amyloidogenesis . The presence of HSPGs in SPs , CAA , and NFTs in AD brains was already demonstrated in the late 1980s -LSB- 63 -- 65 -RSB- . Only when antibodies became available that could identify the various individual HSPG species was it described that perlecan colocalized with all three lesions characteristic of AD brains -LSB- 65 -- 67 -RSB- . However , we were not able to confirm these findings -LSB- 68 , 69 -RSB- . Furthermore , it was shown that in both diffuse and classic SPs , several other HSPGs were found , such as agrin , glypican 1 , and syndecan 1-3 , whereas collagen XVIII is only present in classic SPs and CAA -LSB- 69 -- 72 -RSB- . These data suggest that HSPGs interact with Aβ , thereby contributing to development or persistence of SPs or CAA . HSPGs isolated from Engelbreth -- Holm -- Swarm tumor prevented proteolytic breakdown of aggregated Aβ -LSB- 73 -RSB- . In addition , both agrin and perlecan directly interacted with Aβ and promoted conversion of non-fibrillar Aβ into fibrillar Aβ -LSB- 70 , 74 -- 76 -RSB- . Although the interaction between HSPGs and Aβ is likely mediated predominantly by the sulfate moieties of the GAGs , a role for the protein backbone in Aβ aggregation could not be excluded -LSB- 77 , 78 -RSB- . As sulfated GAGs were also demonstrated in NFTs in AD brains -LSB- 79 -RSB- , these macromolecules may also play a role in tangle development . Indeed , sulfated GAGs may induce the formation of paired helical filaments by stimulating tau phosphorylation -LSB- 80 -RSB- . As heparan sulfates bind to Aβ and interfere with its fibrillogenesis , they are interesting candidates for therapeutic intervention -LSB- 81 -RSB- . GAG mimetics are able to inhibit this binding and may block the formation of β-pleated sheets and adherence of Aβ to the cell surface -LSB- 82 -RSB- . The use of GAG mimetics has already been explored in mouse models where they reduced progression of inflammation-associated amyloidosis -LSB- 83 -RSB- . The efficacy of one of these compounds is currently being tested in a human phase III trial . As exemplified by ApoE , Aβ-binding proteins may play a role in the inflammatory reactions in AD brains . Recently , it was demonstrated that the semi-synthetic proteoglycan analogue dextran sulfate blocks activation of the complement cascade -LSB- 84 -RSB- . In addition , chondroitin sulfate proteoglycans are also known to bind to C1q and prevent the formation of the C1 complex in vitro -LSB- 85 -RSB- . By doing so , chondroitin sulfate proteoglycans inhibit normal complement function . Furthermore , heparin has long been regarded as a potential complement inhibitor -LSB- 86 -RSB- . In conclusion , HSPGs do not only colocalize with Aβ and tau , but they also contribute to the development of these lesions . The role of HSPGs in Aβ aggregation might even be a protective one . HSPGs prevent the persistence of toxic Aβ forms , e.g. , oligomers or protofibrils , and transform them into more harmless aggregates , i.e. , the classic senile plaques containing mature Aβ fibrils that are less toxic than the intermediate aggregates . In addition , HSPGs might play a role in the development of AD lesions by inhibiting complement activation . According to the definitions , HSPGs can therefore be regarded as amateur chaperones . Their ability to recognize a variety of proteins may originate from the heterogeneous structure of the heparan sulfate chains . The negatively charged HS chains are structurally heterogeneous and bind a diverse repertoire of proteins , such as amyloid A , protease-resistant prion protein , α-synuclein , and tau , providing HSPGs with the ability to interact with a wide range of intracellular and extracellular amyloidogenic proteins -LSB- 61 -RSB- . Complement Factors The complement system is an ancient host defense mechanism which is involved in boosting antibody activity . The system consists of a group of soluble serum proteins C1 -- C9 and is activated either by immunoglobulin M or G bound to a foreign particle or directly by microorganisms . Proteins such as Hageman factor , C4 binding protein , CDS46 , CD59 , and C1 inhibitor regulate the complement system . In AD , the complement system is overexpressed and activated -LSB- 46 -RSB- . The Aβ protein itself activates this system , and complement factor concentrations are increased in AD brains -LSB- 87 -- 89 -RSB- . Aβ induces C3 and C4 in AD , and elevated levels of the membrane attack complex -LRB- MAC -RRB- composed of C5 -- C9 have been observed -LSB- 90 , 91 -RSB- . In addition , factors such as Hageman factor , C1q , C3 , and C5 -- 9 are commonly found in SPs and NFTs -LSB- 87 , 92 , 93 -RSB- . C1q is associated with Aβ deposits and directly binds fibrillar Aβ which activates the complement cascade -LSB- 94 -RSB- . In contrast , the complement inhibitor -LRB- C1 INH -RRB- is downregulated in AD -LSB- 95 , 96 -RSB- . Thus , an activated complement system is a general feature observed in AD . However , the contribution of complement to the pathogenesis of AD is controversial . On the one hand , it is suggested that complement activation protects against Aβ-induced toxicity and even contributes to reducing the accumulation of Aβ in SPs -LSB- 97 -RSB- . Transgenic mice expressing complement inhibitors develop increased AD-pathology , whereas increased complement C3 production was associated with a reduction of Aβ deposition -LSB- 97 -RSB- . Thus , the complement activation in the brain may be beneficial in AD and possibly also other neurodegenerative diseases -LSB- 98 -- 100 -RSB- . However , complement activation may lead to accelerated neurodegeneration as well . Activation of complement in an antibody-independent fashion is achieved by binding of aggregated , but not soluble , Aβ to C1q -LSB- 12 , 90 , 101 , 102 -RSB- . This latter finding suggests that in AD , aggregated Aβ induces chronic complement activation . Thus , C1 binding to fibrillar Aβ deposits may precede microglial activation . Both Aβ and pro-inflammatory stimuli are able to activate microglia , which results in increased Aβ and cytokine production -LSB- 103 -RSB- . Furthermore , cultured human microglial cells show an increase in cytokine production after co-stimulation of Aβ with C1q and serum amyloid P -LRB- SAP -RRB- -LSB- 104 -RSB- . This suggests that microglia may get triggered by both Aβ - and SP-associated factors such as C1q , which results in the secretion of pro-inflammatory cytokines and Aβ , both accelerating neurodegeneration . Although none of the complement factors directly regulate conformational changes of Aβ , complement activation as a whole plays a role in the Aβ aggregation in vivo . Therefore , complement factors might act as amateur chaperones , although their exact role in Aβ aggregation remains to be elucidated . Professional Chaperones Heat shock proteins -LRB- Hsp -RRB- are professional chaperones . They are highly conserved proteins constitutively expressed in most cells under normal conditions where they play a role in cellular metabolism and help normal folding processes -LSB- 105 -RSB- . In addition , during cell stress , they bind unfolded proteins to keep them in their native state -LSB- 105 -RSB- . Heat shock proteins can be divided into two different families based on size and function : classic Hsps such as Hsp100 , Hsp90 , Hsp70 , Hsp60 , and the small heat shock proteins -LRB- sHsps -RRB- . Hsps with a molecular weight of 60 kD or more possess an ATP-binding site and are actively involved in the process of refolding of misfolded proteins -LSB- 106 -RSB- . Small Hsps , with a molecular weight of 40 kD or less , lack this ATP-binding site and assist the Hsps in their refolding function -LSB- 107 -RSB- . The role of Hsps in misfolded protein recognition and refolding is illustrated in Fig. 1 . Fig. 1The role of heat shock proteins -LRB- Hsp -RRB- and small heat shock proteins -LRB- sHsps -RRB- in recognition and refolding of unfolded and misfolded proteins . Unfolded or misfolded proteins are recognized by Hsps and sHsps . Together with these unfolded or misfolded proteins , Hsps and sHsps form a complex . In addition , Hsps recover unfolded or misfolded proteins back to their native form using ATP . If unfolded or misfolded protein are not recognized by the Hsp/sHsps , these unfolded or misfolded proteins are capable of forming aggregates Small Heat Shock Proteins Small Hsps function as molecular chaperones that can prevent proteins from adopting an incorrect conformation -LSB- 108 -RSB- . The sHsp family is characterized by the presence of an α-crystallin domain , a stretch of 80 -- 100 amino acids in the C terminal half of the proteins -LSB- 109 -RSB- . So far , the sHsp family comprises ten sHsps , including αB-crystallin , Hsp27 , Hsp20 , HspB8 , and HspB2/B3 -LSB- 110 -RSB- . Although sHsps are predominantly expressed in muscle cells , several family members are also found in human brain . In AD , αB-crystallin and Hsp27 are upregulated and expressed by astrocytes surrounding SPs and NFTs -LSB- 111 -- 114 -RSB- , whereas Hsp20 , HspB2 , and HspB8 colocalize with Aβ in SPs and CAA -LSB- 115 , 116 -RSB- . Although αB-crystallin or Hsp27 do not colocalize with Aβ in SPs , direct interaction between Aβ and these sHsps in addition to Hsp20 and HspB8 has been demonstrated -LSB- 111 , 117 -- 119 -RSB- . In addition , high-affinity binding of αB-crystallin and Aβ has been observed in eye lenses from AD patients -LSB- 120 -RSB- . Furthermore , αB-crystallin is able to prevent mature Aβ fibril formation , retaining it in a non-fibrillar , but likely a protofibrillar state , which is highly toxic to neurons -LSB- 121 -RSB- . Recently , we demonstrated that αB-crystallin , Hsp20 , and HspB8 inhibit Aβ-mediated toxicity towards cerebrovascular cells probably by preventing aggregation of Aβ at the cell surface -LSB- 116 , 117 -RSB- . Others showed that Hsp27 directly binds to hyperphosphorylated tau , thereby protecting against cell death -LSB- 122 -RSB- . Hsps are involved in the formation and persistence of misfolded protein aggregates . They are upregulated in several neurodegenerative diseases , such as AD , Creutzfeldt -- Jakob disease , and Parkinson 's disease probably as a reaction to the formation of misfolded proteins -LSB- 113 , 123 -- 126 -RSB- . However , despite of increased intracellular levels , they are unable to prevent accumulation of Aβ in AD possibly because of decreased chaperone activity . In aged rats , this was illustrated by a significant decrease of Hsp90 function -LSB- 127 -RSB- , resulting in diminished hepatic chaperone capacity . Furthermore , the increasing amount of damaged or misfolded proteins as a result of defects in protein degradation might lead to a total decrease in chaperone activity in aged cells -LSB- 128 -RSB- . Thus , the state of misfolded protein recognition and repair systems , such as the -LRB- s -RRB- Hsp system , might be of great importance in the development of neurodegenerative diseases . Miscellaneous Proteins Apart from the above-described proteins , several other molecules are also associated with the pathological lesions of AD , and some of these can be regarded as amateur chaperones . Acute phase proteins , such as α1-antichymotrypsin -LRB- ACT -RRB- , α2-macroglobulin -LRB- α2M -RRB- , and SAP , are all associated with Aβ deposition -LSB- 129 -- 132 -RSB- . ACT is a serine protease inhibitor of the serpin family , and in AD , ACT levels are upregulated , and binding of ACT with Aβ induces Aβ fibrillogenesis -LSB- 133 -- 135 -RSB- . Furthermore , when ACT is overexpressed in transgenic mice , an increased plaque load in the brains of these mice and impaired spatial learning is observed -LSB- 134 , 135 -RSB- . α2M also binds Aβ , although in contrast to ACT , this binding prevents Aβ fibril formation and fibril-associated neurotoxicity -LSB- 136 , 137 -RSB- . α2M promotes the protease-mediated degradation of α2M/Aβ complexes and contributes to clearance of Aβ from the brain -LRB- discussed in paragraph 4 -RRB- -LSB- 138 , 139 -RSB- . The glycoprotein SAP belongs to the pentraxin family and is a common component of all known types of amyloid fibrils . SAP is upregulated in AD and protects amyloid fibrils from proteolysis in vitro -LSB- 140 , 141 -RSB- . SAP not only colocalizes with SPs and interacts with aggregated Aβ ; SAP oligomers also bind and activate C1 -LSB- 142 -RSB- . Both C1 and SAP may bind to fibrillar Aβ deposits in vivo and induce microglial activation , as cultured human microglial cells show an increase in cytokine production after co-stimulation of Aβ with C1q and SAP -LSB- 104 -RSB- . These observations further strengthen the above-noted suggestion that not only Aβ , but also several Aβ-binding proteins , are capable of activating the complement system , and thus , contribute to neuroinflammation in AD . In addition , both α2M and ACT , in contrast to SAP , can be regarded as amateur chaperones , as they regulate conformational changes of Aβ . Tissue-type plasminogen activator -LRB- tPA -RRB- regulates activation of plasminogen into plasmin and is expressed in various regions of the brain especially in the hippocampus -LSB- 143 -RSB- . Several reports suggested an important role for tPA in AD , as the tPA system is involved in Aβ turnover -LSB- 144 , 145 -RSB- . Fibrillar forms of Aβ stimulate tPA activity in vitro , whereas in AD patients , a reduction of tPA activity is observed in the affected areas -LSB- 144 , 146 -RSB- . Although tPA has no effect on conformational changes of Aβ , it might play a role in the clearance of Aβ from the brain -LRB- paragraph 4 -RSB- . The actin-regulatory protein gelsolin is found both intracellularly and in plasma -LSB- 147 , 148 -RSB- . Plasma gelsolin can be considered an amateur chaperone , as it binds Aβ and not only inhibits its Aβ fibrillization but is also capable of degrading preformed Aβ fibrils -LSB- 149 , 150 -RSB- . Furthermore , gelsolin inhibits Aβ-mediated neurotoxicity -LSB- 151 -RSB- . One of the major gangliosides in the brain is GM1 . Soluble Aβ binds GM1 and the formed complexes accelerate Aβ fibrillogenesis by acting as a seed for Aβ -LSB- 152 -RSB- . In the presence of GM1 , Aβ is more neurotoxic than Aβ alone , and cholesterol-rich membranes demonstrate accelerated Aβ binding due to the formation of GM1 clusters -LSB- 153 , 154 -RSB- . As GM1 is a major component of lipid rafts and recent studies suggest that Aβ accumulation in these lipid rafts is an early event in AD development , GM1 might play an important role in the early steps in AD pathogenesis -LSB- 155 , 156 -RSB- . In summary , several proteins are associated with Aβ aggregates in the AD brain and contribute to the aggregation of Aβ and should , therefore , be considered as amateur chaperones . In addition , they might play a role in triggering inflammation . Aβ-Binding Proteins and Intracellular Interactions with Aβ Intracellular accumulation of Aβ already starts in the ER or in the Golgi apparatus of the cell -LSB- 157 -- 159 -RSB- . Intracellular Aβ is associated with neuronal damage -LSB- 160 , 161 -RSB- , and intraneuronal accumulation of Aβ in transgenic mice was correlated with impairments in synaptic plasticity -LSB- 162 -RSB- . Intraneuronal accumulation of Aβ in those brain areas affected earliest in AD suggests a possible relation between intracellular Aβ and development of AD -LSB- 160 -RSB- . A few proteins that interact with intracellular Aβ and affect its intracellular fate have been identified . The endoplasmic reticulum amyloid beta-peptide-binding protein binds intracellular Aβ and mediates neurotoxicity in neuronal cells by forming an intracellular target for Aβ -LSB- 163 -RSB- . In addition , the mitochondrial enzyme amyloid-β alcohol dehydrogenase also binds Aβ inside neurons , resulting in the production of free radicals -LSB- 164 -RSB- . However , whether these intracellular Aβ-binding proteins affect aggregation of Aβ within the cells remains unknown . Therefore , both these proteins can not , for the time being , be defined as amateur chaperones of Aβ . The first lines of defense against misfolded and aggregated proteins are the professional chaperones , which counteract these processes and are able to stimulate clearance of misfolded proteins by proteosomal degradation . Newly synthesized proteins are folded by several other proteins , such as immunoglobulin-binding protein -LRB- BiP -RRB- / glucose-regulated protein -LRB- GRP78 -RRB- , and calnexin . GRP78 is a member of the Hsp70 protein family and interacts with intracellular APP . GRP78 regulates APP and Aβ secretion by intervening between APP and β - / γ-secretases within the cell -LSB- 165 -RSB- . It is not surprising that in AD , where misfolded protein molecules accumulate , both Hsp90 and Hsp70 synthesis is increased . Several members of the Hsp family directly interact with intracellular Aβ , but only recently , Hsp70 was identified as a protector against intracellular Aβ accumulation -LSB- 166 , 167 -RSB- . Besides , immunoreactivity of both Hsp90 , 70 , and Hsp60 is found in SPs -LSB- 132 -RSB- , which suggests that these professional chaperones may not only interact with misfolded protein in the cell interior -LSB- 168 -- 171 -RSB- . In addition , it has also been postulated that up-regulation of Hsp90 and Hsp70 may suppress the formation of NFTs by partitioning tau into a productive folding pathway and thereby preventing its aggregation -LSB- 172 -RSB- . Recently , it was demonstrated that the chaperone CHIP -- Hsc70 complex conjugates ubiquitin to hyperphosphorylated tau , which enhances cell survival by elimination of soluble hyperphosphorylated tau -LSB- 173 -RSB- . These data suggest that the cell increases production of the Hsps to cope with the presence of misfolded proteins such as hyperphosphorylated tau and accumulating Aβ . At some point , this protective mechanism seems to fail , however . In line with this hypothesis , it was shown that the actin and tubulin specific chaperone Hsp60 is decreased in AD , resulting in a decrease of cytoskeletal proteins in AD-affected neurons -LSB- 174 -RSB- . Thus , both production and function of Hsps seems to be disturbed in AD , which might result in the accumulation of misfolded proteins . The role of other Hsps in regulating intracellular Aβ or tau folding remains to be investigated -LRB- Table 1 -RRB- . Table 1Summary of the expression of chaperones in AD brains and their interaction and effects on Aβ and tau SP/CAANFTDirect interactionEffects on Aβ or tau in generalApolipoproteins ApoE + + Aβ/tau ↑ Fibrillar Aβ / ↓ hyperph . Tau ApoJ + ? Aβ ↓ Aβ aggregationHSPGs Perlecan ± ± AβHSPGs : Agrin + − Aβ ↓ Proteolytic breakdown Aβ Glypican 1 + − ? ↑ Non-fibrillar → fibrillar Aβ Syndecan 1 -- 3 + − ? ↑ Phosphorylation tau Collagen XVIII + − ? GAGs + + Aβ/tauComplement factors Hageman Factor + + ? Aβ activates complement in AD C1q + + AβC3 ↓ Aβ deposition C3/C4 + + Aβ C5-9 + + ? Heat shock proteins Hsp90 + ? Tau ↓ Tau aggregation Hsp70 + ? Aβ/tau ↓ Tau aggregationSmall Hsps αB-crystallin − − Aβ ↓ Aβ fibril formation Hsp27 − ± Aβ/tau ↓ Aβ fibril formation Hsp20 + − Aβ ↓ Aβ fibril formation HspB2/B3 + + -- No effect HspB8 + − Aβ ↓ Aβ fibril formationAcute phase proteins α1-antichymotrypsin + − Aβ ↑ Aβ fibrillization α2-macroglobulin + − Aβ serum amyloid P + + Aβ ↑ Aβ fibrillizationMiscellaneous compounds tPA − − Aβ ↓ Aβ fibril formation Gelsolin − − Aβ ↑ Aβ fibrillizationExpression of chaperones in a specific lesion is illustrated as follows : present -LRB- + -RRB- , by conflicting reports -LRB- ± -RRB- , absence -LRB- − -RRB- , and unknown -LRB- ? -RRB- ; ↓ = inhibition or down-regulation , ↑ = induction or up-regulationSP Senile plaques , CAA cerebral amyloid angiopathy , NFT neurofibrillary tangles , HSPGs heparan sulphate proteoglycans , Aβ amyloid-beta , Hsp heat shock proteins , Apo apolipoproteins , SAP serum amyloid P , tPA tissue-type plasminogen activator , GAGs glycosaminoglycans , LDLR low-density lipoprotein receptor , LRP-1 LDL receptor protein-1 , BBB blood -- brain barrier Aβ-Binding Proteins and Aβ Clearance Aβ-binding proteins , amateur chaperones , play a role in the clearance of Aβ from brain by functioning as a transporter molecule . Two major pathways govern Aβ clearance . By the first pathway , Aβ is removed from brain to blood via active transport across the BBB . This active transport is performed by specialized transporters , so-called `` Aβ-receptors '' , expressed by endothelial cells . Second , Aβ is removed from brain via phagocytosis by both microglial cells and astrocytes . In both pathways , interaction of Aβ with cell surface Aβ-receptors is crucial ; therefore , the expression levels of Aβ-binding proteins might contribute to Aβ clearance by regulating its binding with Aβ receptors . The low-density lipoprotein receptor-related protein-1 -LRB- LRP-1 -RRB- binds Aβ in a complex with ApoE at the abluminal side of the endothelium and internalizes these ApoE/Aβ complexes followed by degradation in lysosomes or transport into the plasma -LSB- 4 , 175 -RSB- . However , LRP-1 also mediates transport of free Aβ across the BBB -LSB- 10 -RSB- . In contrast to LRP-1 , the receptor for advanced glycation end products -LRB- RAGE -RRB- transports Aβ from the circulation into the central nervous system -LSB- 176 -RSB- . Similar to RAGE , the Aβ receptor megalin is also involved in the transport of Aβ from blood to brain , although megalin probably plays only a minor role in Aβ transport . Furthermore , megalin binds Aβ/ApoE complexes rather than free Aβ -LSB- 177 , 178 -RSB- . Clearance of Aβ/ApoE complexes from brain might be ApoE isoform-dependent . ApoE4 forms less stable complexes with Aβ than ApoE3 or ApoE2 ; therefore , ApoE4 reduces Aβ transport efficiency across the BBB . Additionally , as described above -LRB- paragraph 2 -RRB- , ApoE4 enhances Aβ aggregation more efficiently than ApoE3 , which also inhibits clearance . On the other hand , the LDL receptor shows a marked preference for the ApoE3 and ApoE4 isoforms and binds the ApoE2 isoform poorly -LSB- 179 -RSB- . Given the similarity between the LDL receptor family , other LDL receptors , such as the LRP-1 receptor , might display similar specificities towards the ApoE isoforms , but this has not been reported yet . Moreover , lipidation of ApoE also affects clearance of ApoE and ApoE/Aβ complexes from brain , as LRP preferentially binds lipid-rich forms of ApoE -LSB- 179 -RSB- . These data indicate that Aβ-binding proteins , especially ApoE , and possibly , ApoJ , play an important role in transport of Aβ across the BBB and that both the ApoE isoform and the ApoE lipidation state affect Aβ clearance . In addition to ApoE and ApoJ , the Aβ-binding protein α2M also forms complexes with Aβ . As α2M is a ligand for LRP-1 , these α2M/Aβ complexes might undergo LRP-1-mediated endocytosis and degradation or translocation into the plasma -LSB- 7 , 139 -RSB- . Stimulation of the transport of Aβ across the BBB demonstrated to be an effective therapeutic approach in AD , as several studies demonstrated elevated levels of Aβ in the plasma of mice after passive immunization with anti-Aβ antibodies or Fab fragments -LSB- 180 -- 182 -RSB- , and decline in cognitive performance was arrested in patients that received vaccination -LSB- 183 -RSB- . However , the occurrence of severe meningoencephalitis in human patients after active immunization with Aβ hampered widespread application of this type of therapy . Administration of Aβ-binding proteins that demonstrate similar positive effects , but possibly , without the severe immune reactions associated with antibody therapy , might provide an alternative strategy . An interesting example of such an Aβ-binding protein is gelsolin . This protein has high affinity for Aβ and reduces Aβ levels in a transgenic mouse model of AD -LSB- 184 -RSB- . Furthermore , administration of gelsolin or GM1 in PS/APP mice resulted in decreased Aβ aggregation in the brains -LSB- 184 -RSB- . Both gelsolin and GM1 act as a `` peripheral sink '' for Aβ . Although both compounds did not enter the brain , they lowered soluble Aβ concentrations in the blood , shifted the balance between blood and cerebral Aβ concentrations , and accordingly , stimulated Aβ transport over the BBB . Therefore , other Aβ-binding proteins administered in the circulation might also act as `` peripheral sinks '' -LSB- 181 , 184 -RSB- . Both activated microglial cells and activated astrocytes are associated with Aβ deposition and may internalize Aβ fragments via phagocytosis -LSB- 185 -- 187 -RSB- . Activation of the complement system is , among others , achieved by Hsps such as Hsp60 and Hsp70 , which are able to induce phagocytosis by microglia , and thus , clearance of Aβ -LSB- 132 , 188 , 189 -RSB- . In addition , the absence of ApoE reduces internalization and degradation of Aβ by astrocytes in the brain , demonstrating that ApoE is directly involved in the clearance of Aβ from brain via phagocytosis by microglial cells and astrocytes -LSB- 40 -RSB- . tPA might also contribute to clearance of Aβ , as it accelerates Aβ clearance from transgenic mouse brains -LSB- 146 -RSB- . Thus , as Aβ-chaperones contribute to activation of the complement system or activation of microglial cells and astrocytes , these proteins might contribute to the clearance of Aβ from the brain via phagocytosis . Concluding Remarks Professional chaperones , such as the heat shock protein family , and amateur chaperones , such as apolipoproteins and HSPGs and several other proteins , have a role in the intracellular handling of misfolded proteins , induce conformational changes of proteins , or act as transporter of proteins -LRB- Fig. 2 -RRB- . This suggests that these chaperones form interesting therapeutic targets in the prevention and treatment of neurodegenerative diseases . Fig. 2The putative role of chaperones in amyloid-β -LRB- Aβ -RRB- fibril formation , proteolytic breakdown , and clearance from the brain . In Alzheimer 's disease , soluble Aβ , predominantly produced in neurons , is converted into β-sheet rich protofibrils and eventually forms mature Aβ fibrils . The conversion from soluble Aβ to protofibrils and fibrils , which accumulate in senile plaques and cerebral amyloid angiopathy , is enhanced by chaperones as apolipoprotein E -LRB- ApoE -RRB- , Gelsolin , α1-antichymotrypsin -LRB- ACT -RRB- and several heparan sulphate proteoglycans -LRB- HSPGs -RRB- , which function as catalysts . In contrast , the heat shock protein family , tissue-type plasminogen activator -LRB- tPA -RRB- and complement factors prevent the transition of soluble Aβ into protofibrils and mature fibrils . Furthermore , heat shock proteins and tPA stimulate the proteolytic breakdown of -LRB- proto -RRB- fibrils , whereas HSPGs prevent this breakdown . Finally , the clearance of Aβ from the brain across the blood -- brain barrier is stimulated by ApoE , ApoJ , and α2-macroglobulin -LRB- α2M -RRB- , whereas complement factors stimulate phagocytosis-mediated clearance of Aβ by activated microglia and astrocytes In the process of clearance of Aβ from the brain , Aβ-binding partners might play important roles by acting as Aβ transporter proteins in both the receptor-mediated clearance of Aβ across the BBB but also as a `` peripheral sink '' for Aβ . Both ApoE isotype and local concentrations in the brain might regulate Aβ transport across the BBB , but as this transport is receptor-mediated , other Aβ-binding proteins might also fulfill such a role . In addition , transport of aggregated Aβ across the BBB is less efficient than soluble Aβ . Thus , by preventing self-aggregation of Aβ , Aβ-binding proteins contribute to the clearance of Aβ from the brain . As a therapeutic strategy , Aβ-binding proteins serving as a `` peripheral sink '' , such as gelsolin , seem promising -LSB- 184 -RSB- . Overexpression of professional chaperones , such as the Hsps , to prevent aggregation of misfolded proteins will have to be evaluated carefully , as they also interact with other chaperones and are dependent on this interaction to fulfill some of their functions . This strategy may therefore result in instability of the cell-stress mechanism , which may cause the system to collapse . A solution may be found in the overexpression of several chaperones , which may be required to achieve an impact on the progression of the disease . Another pitfall in the use of professional chaperones as therapeutic agents is their ability to bind misfolded proteins and keep them in an intermediate conformation . This type of conformation might even be more toxic than the aggregated state . As an example , co-incubations of αB-cystallin with Aβ are more toxic to neurons than Aβ alone -LSB- 121 -RSB- . Furthermore , Hsps are most likely to be involved in early development of neurodegenerative diseases , given their natural function . Yet , the role of this protein family in maturation of the neurodegenerative lesions remains to be elucidated . In conclusion , studying the role of chaperones , both professional and amateur , in the pathophysiology of AD will provide us with a better understanding of the mechanisms underlying the formation and accumulation of toxic aggregates in AD , which , eventually , will lead to the design of more effective therapeutic strategies .

B I I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O B I I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B B I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I I O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

amyloid
heat shock proteins
heparan sulfate proteoglycans
apolipoproteins
chaperones

alzheimer’s disease
amyloid beta-protein