Patent Application: US-18847905-A

Abstract:
ig - positive neurons , which have been shown to be present in alzheimer &# 39 ; s disease , are shown to have complement c1q and c5b - 9 proteins . c1q and c5b - 9 can be employed in diagnosis and treatment of alzheimer &# 39 ; s disease .

Description:
postmortem entorhinal cortical brain tissues from patients with sporadic ad ( n = 12 ) and age - matched control ( n = 6 ) were obtained from the harvard brain tissue resource center ( hbtrc , belmont , mass ., usa ) and fixed in 10 % neutral - buffered formalin . pathological confirmation of ad included ( 1 ) the presence of amyloid plaques , ( 2 ) the presence of neurofibrillary tangles and ( 3 ) reduced neuronal density ( 1 , 5 , 6 ). tissues were trimmed and processed for paraffin embedding according to conventional methods . five - micron sections were serially cut , mounted onto superfrost plus + ( fisher scientific , pittsburgh , pa .) microscopic slides and dried overnight . the protocols for routine single immunohistochemistry ( ihc ) have been described in detail previously ( 1 , 5 , 6 ). briefly , tissue sections on microscopic slides were dewaxed and re - hydrated . slides were microwaved in target buffer ( dako , carpenturia , calif . ), cooled , placed in phosphate - buffered saline ( ph 7 . 4 , pbs ) and treated with 3 . 0 % h 2 o 2 for 10 min at room temperature . all incubations ( 30 min each ) and washes were performed at room temperature . normal blocking serum ( vector labs , burlingame , calif .) was placed on all slides for 10 min . after a brief rinse in pbs , sections were treated with the primary antibodies : polyclonal anti - human pan - ig recognizes igg , igm , iga ( 1 : 1000 , serotec , n . c . ; dako , calif . ); polyclonal anti - human c1q ( 1 : 200 , quidel , calif . ); monoclonal anti - human c5b - 9 ( 1 : 200 , quidel , calif . ); and monoclonal anti - human hla - dr , a marker of reactive microglia ( 3 , 4 , 6 , 7 , 8 ) ( 1 : 200 , chemicon , temecula , calif .). slides were then washed in pbs and treated with goat anti - rabbit ( polyclonal primaries ) or horse anti - mouse ( monoclonal primaries ) biotinylated secondary antibodies ( vector labs ). after washing in pbs , the avidin - biotin - horseradish peroxidase complex reagent ( abchp , vector labs ) was added . all slides were washed and treated with 3 , 3 ′- diaminobenzidine ( dab , biomeda , foster city , calif .) 2 times 5 min , rinsed in distilled water , and counterstained with hematoxylin . in addition , to determine if there was a spatial ( two - dimensional ) association between the reactive microglia and the ig - positive neurons , the distance ( μms ) between the nuclei of ig - positive or ig - negative neurons and prominent , red - labeled , hla - dr immunoreactive microglial fibers were measured using image analysis ( image pro , ver . 4 . 01 , phase iii imaging , glen mills , pa .). to accomplish this objective , double ihc was applied . briefly , the first primary antibody was detected using the abc - alkaline phosphotase ( abcap ) reagent followed by the fast red ( sigma , mo .) chromogen , while the second primary antibody ( pan - ig ) was detected using the abchp - dab system ( 1 , 6 , 8 ). negative controls included replacement of the primary antibody with the antibody diluent or pre - absorption of the primary antibody with its specific antigen as previously demonstrated for the ig antibodies ( 1 ). ig - positive and ig - negative neurons ( average 20 / tissue ) were analyzed in 10 random fields ( 40 × objective )/ tissue ( n = 5 ). statistical analyses were performed using the mann - whitney rank sum test using sigmastat and plotted using sigmaplot . representative brown labeled , ig -, c1q - and c5b - 9 - immunopositive neurons ( arrows ) were detected in the entorhinal cortex of ad brain tissues ( fig1 ). not detectable immunolabeling was observed in the negative controls ( data not presented ). the panels in fig1 a show the presence of intensely immunolabeled ig - positive neurons ( arrows ) within close proximity ( within microns ) of ig - negative neurons ( arrowheads ), which had also been previously reported ( 1 , 2 ). note the neurodegenerative features such as cell atrophy and dense , pyknotic nuclear chromatin ( fig1 a , insert ) of the ig - positive neurons ( arrows , insert ) amidst normally appearing ig - negative neurons with prominent nucleoli embedded in normally appearing , transcriptional active nuclear euchromatin ( arrowheads ), which was also previously described ( 1 , 2 ). the panels in fig1 b show prominent c1q - immunopositive neurons ( arrows ) amidst c1q - negative neurons ( arrowheads ). this labeling was primarily located in the neuronal perikaryon with diminished signal in the somatic - dendritic area . it was interesting to note that these c1q - positive neurons exhibited the same neurodegenerative features as the ig - positive neurons with the atrophic perikaryon and dense , pyknotic nuclear chromatin ( fig1 b , insert ) suggesting not only the c1q - positive neurons were degenerative but that the ig and c1q immunolabeling patterns may be co - localized in neurons . weak c1q immunolabeling was also detected in some morphologically healthy neurons indicating basal expression detection , as the expression of c1q in another study was low in naïve mice but increased in experimental autoimmune encephalomyelitis ( eae ) ( 9 ). similar findings were also observed in the c5b - 9 - immunopositive neurons ( arrows , fig1 c ). again , only a small population of the neurons was c5b - 9 immunolabeled , some of which appeared neurodegenerative ( fig1 c , insert ). arrowheads in fig1 c show the lack of c5b - 9 detection in neighboring neurons . even though the labeling patterns of the ig , c1q and c5b - 9 appeared to be associated with neurodegenerative neurons , it was not clear if they were also present in the same cell until we analyzed serially ( 5 μs ) sectioned labeled tissues . these data showed that c1q and c5b - 9 are indeed present in ig - positive neurons ( fig2 ). specifically , in representative serial sets , fig2 a - c showed c1q immunoreactivity ( fig2 a - c ) in the ig - positive neurons . c5b - 9 immunoreactivity was also detected in the ig - positive neurons ( fig2 d ). however , in this particular example , relatively weaker ig immunolabeling was detected ( fig2 d insert ) in the c5b - 9 neuron , which may suggest the lost of ig immunoreactivity as the cell nears death . importantly , the data obtained from these assays also showed that ig - positive neurons are neurodegenerative and are c1q and c5b - 9 immunopositive suggesting a strong association between these signatures or profiles . as noted , age - matched control brains were also analyzed for the presence of ig , c1q and c5b - 9 . although parenchymal ig immunolabeling was detected in these tissues , most of the labeling was restricted around large vessels ( data not presented ), as previously described ( 1 ). several ig - positive neurons were only observed in one of the six age - matched control brain tissues ( data not presented ), which also showed c1q and c5b - 9 immunolabeling patterns . c1q , a classical complement pathway component ( 3 , 10 ) ( fig1 b ), and c5b - 9 , a marker of the terminal step in the complement pathway and representing the membrane attack complex ( 4 , 10 , 11 ) ( fig1 c ) were detected in ig - positive neurons ( fig2 ) providing evidence for the presence of the classical , antibody - dependent ( not alternative , antibody - independent ) complement pathway of cell death to the ig - positive neurons . immunocytochemical studies have demonstrated that the complement cascade is fully activated in the ad hippocampus and neocortex resulting in ‘ deposition ’ of c5b - 9 on dystrophic neuritis , nfts and senile plaques ( 12 , 13 ), and now within ig - positive neuronal perikaryon . the significance of intracellular ig , c1q and c5b - 9 is not known but may represent internalization as well as de novo synthesis ( 14 ). as a note , complement activation products have been described as deposition material ( 15 , 16 ). however , any evidence of ‘ deposition ’ from a random point of view was not observed . to clarify , any detection of complement components were associated with cells in this study . next , a series of assays were performed to characterize the spatial distribution of the reactive microglia ( red - labeled ) with ig - positive neurons ( brown - labeled ) using double ihc on tissue sections ( fig3 ). in fig3 a - e , reactive microglia are located within very close proximity ( possibly in contact ) to many ig - positive neurons ( arrows ), which were not readily observed nearby ig - negative neurons ( arrowheads ). although subject to over interpretation , the microglia detected in fig3 a may represent an early stage of discovery as the ig - positive neuron appears morphologically healthy , as compared to the microglia detected in fig3 panels b - d appear in contact with the ig - positive neuronal perikaryon that appear slightly dystrophic . the arrangement of the microglia and ig - positive neurons in fig3 e - g resemble that of a war zone ( late stage ) where several red - labeled , reactive microglia appear dramatically engaged with the degenerative ig - positive neurons ( fig3 e , f , g ). note the lack of associated reactive microglia with the ig - negative neurons ( arrowheads , fig3 ). in an effort to determine if the reactive microglia target ig - positive neurons over the ig - negative cells , the average distances between the hla - dr - positive processes of the microglia to the ig - positive and ig - negative neuronal nuclei was measured using image analysis . as presented in fig4 , the analytical data showed that the reactive microglia are significantly ( p & lt ; 0 . 001 ) more associated with the ig - positive neurons than the ig - negative neurons . although these data were obtained from static , two - dimensional images , it is difficult to ignore the possibility that reactive microglia are more associated with the ig - positive ( n = 101 ; mean = 7 . 69 μm ± 0 . 72 se ) that the ig - negative ( n = 172 ; mean = 31 . 74 μm ± 1 . 44 se ) neurons . these data suggest the involvement of microglia in the pending cell death process . in support , c1q and c5b - 9 can bind to the surface of apoptotic cells resulting in the phagocytosis of these cells by microglia ( 17 , 18 ). also , complement produced locally by reactive microglia was activated on the membranes of neurons in huntington &# 39 ; s disease contributing to neuronal necrosis as well as proinflammatory activities ( 14 ). hence , it is logical to explain the presence of these microglia on these ig - positive complement - ridden neurons , but it remains to be determined if the glial cells are recruited to these neurons or if they contribute to complement accumulation on these neurons . however , a recent study suggested the former to be true , as there was significantly less microglial activation surrounding fibrillar aβ deposits in the c1q null mouse ( 7 ). previously , it was proposed that the presence of the antibody - induced classical complement pathway did not exist in ad because the discovery of an antibody remained “ unequivocally demonstrated ” in spite of overwhelming evidence of classical pathway components and activation fragments reported in the ad brain ( 13 , 19 , 20 ). subsequently , β - amyloid as well as other neuropathological markers had been proposed to be responsible for complement activation ( 21 ). as a personal observation , aβ42 was detected in ig - positive and ig - negative neurons suggesting that ig immunoreactivity was independent of aβ42 immunoreactivity in neurons ( data not presented ). an autoimmune ad hypothesis based upon the dramatic increases of vascular - derived , parenchymal igs and ig - positive , neurodegenerative neurons had been suggested ( 1 , 2 ). the data from the present study validates and extends the proposed pathway ( 1 , 2 ) which most likely begins with a dysfunctional , unregulated , bbb that allows non - discriminatory passage of vascular derived - ig into the brain parenchyma . once in the cns , some of these once benign antibodies , inconsequentially bind to their neuronal or neuronal - like antigen ( s ) on neurons leading to complement formation ( mac ) resulting in an autoimmune / classical ( antibody - dependent ) complement - cell death process in the ad brain . these observations are not limited to ad , as the activation of the classical complement system is also known to play a central role in autoimmune demylineation ( 9 ), and in multiple sclerosis , myasthenia gravis , head trauma and stroke ( 22 ). although not clear , the ability or capacity of these vascular derived antibodies to specifically or non - specifically bind to their neuronal target may able be dependent upon its isotype , avidity and affinity ( 1 , 2 ) as well as its ability to fix complement . each factor will play an important role in determining the ‘ clinical ’ pathogenicity of an autoantibody response to its antigen ( 20 ) and may explain why bbb breach alone , or that the presence of parenchymal igs may not always lead to ad . regardless , the effects of the “ inconsequential ” binding of these antibodies to a specific population of neurons did not appear favorable . the data suggests that populations of ig - positive neurons are dying via the classical complement pathway and that microglia are preferentially associated with many of these degenerating neurons . unfortunately , once the 2 - hit ( presence of specific neuronal damaging antibodies and bbb breach ) cascade pathway begins , subsequent processes of inflammation could inflict additional neuronal cell death ( 23 ) independent of immunoglobulin and complement . as it was previously noted ( 1 , 2 ), the presence of this “ auto ”- antibody , once characterized , should provide a new therapeutic target to treat and possibly prevent ad . in the meantime , therapeutic opportunities should be designed to preserve the integrity of the bbb in an effort to block the anomalous presence and subsequent deleterious actions of autoantibody ( s ) into the cns , while other strategies could be directed to augment , remove or block the autoantibody while it is in the vascular system before it gains circuitous entry into the cns . minimally , in the context of this work , cns imaging data to assess bbb integrity “ coupled ” with the presence of vascular disease indicators and autoimmune products ( i . e . complement ) could provide diagnostic and prognostic capabilities , in addition to the clinical cognitive testing paradigms . 1 . d &# 39 ; andrea m r : evidence linking neuronal cell death to autoimmunity in alzheimer &# 39 ; s disease . brain research 2003 ; 982 : 19 - 30 . 2 . d &# 39 ; andrea mr : add alzheimer &# 39 ; s disease to the list of autoimmune diseases . medical hypotheses 2005 ; 64 ( 3 ): 458 - 463 . 3 . emmerling m r , watson m d , ruby c a , spiegel k : the role of complement in alzheimer &# 39 ; s disease pathology . biochem biophys acta 2000 ; 1502 : 158 - 171 . 4 . webster s , lue l f , brachova l , tenner a j , mcgeer p l , teria k , walker d g , bradt b , cooper n r , rogers j : molecular and cellular characterization in the membrane attack complex , c5b - 9 , in alzheimer &# 39 ; s disease . neurobiology of aging 1997 ; 18 : 415 - 421 . 5 . d &# 39 ; andrea mr , nagele r g , wang h - y , peterson p a , lee d h s : evidence that neurons accumulating amyloid can undergo lysis to form amyloid plaques in alzheimer &# 39 ; s disease . histopathology 2001 ; 38 : 120 - 134 . 6 . d &# 39 ; andrea m r , reiser p a , gumula n a , hertzog b m , andrade - gordon p : application of triple - label immunohistochemistry to characterize inflammation in alzheimer &# 39 ; s disease brains . biotechnic & amp ; histochemistry 2001 ; 76 : 97 - 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