Patent Application: US-36506203-A

Abstract:
the invention provides transgenic , non - human animals and transgenic non - human mammalian cells harboring a transgene encoding a p25 polypeptide . the two neuropathological lesions associated with alzheimer &# 39 ; s disease are amyloid plaques and neurofibrillary tangles , composed predominantly of amyloid β peptides and hyperphosphorylated tau , respectively . while animal models for plaque formation exist , there is no animal model that recapitulates the formation of nfts . this invention provides transgenic mice that overexpress human p25 , an activator of cdk5 , resulting in tau that is hyperphosphorylated at ad - relevant epitopes . deposition of tau is detected in the amygdala , thalamus and cortex . increased phosphorylated neurofilament , silver - positive neurons and neuronal death are also observed in these regions . we conclude that the overexpression of p25 , an activator of cdk5 , is sufficient to produce hyperphosphorylation of tau and neuronal death . the p25 transgenic mouse represents the first model for tau pathology in ad .

Description:
generally , the nomenclature used herein and the laboratory procedures in cell culture , molecular genetics , nucleic acid chemistry and hybridization , biochemistry , histology and immunocytochemistry described below are those well known and commonly described in the art . standard techniques are used for recombinant nucleic acid methods , polynucleotide synthesis , cell culture , transgene incorporation , western blotting , immunocytochemistry and histological techniques such as silver staining . the techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided throughout this specification . the procedures therein are well known in the art and are provided for the convenience of the reader . all the information contained therein is incorporated herein by reference . in accordance with the foregoing objects , in one aspect of the invention are provided nonhuman animals harboring at least one copy of a transgene comprising a polynucleotide sequence that encodes a heterologous protein operably linked to regulatory elements that are capable of expressing the heterologous protein in the transgenic nonhuman animal . said heterologous polypeptide is a truncated portion of the p35 cyclin dependent kinase 5 ( cdk5 ) regulatory protein ( 27 , 28 ). the truncated heterologous polypeptide has the first 98 n - terminal amino acids removed and has a molecular weight of approximately 25 , 000 daltons . this novel polypeptide will be referred to as p25 . typically , the nonhuman transgenic animal is a mouse and the heterologous gene is the human p25 sequence . transgenes are typically cdna sequences that have been operably linked to cis - acting regulatory sequences that direct expression in the host transgenic mammal in cell - type specific manner , typically in neurons . typically , the transgene will be incorporated into the host chromosomes in random , non - targeted fashion . the invention further provides that the nonhuman , transgenic animal harboring at least one copy of the heterologous p25 sequence transgene or gene targeting vector of the invention either non - homologously or homologously integrated into the chromosomal location express the p25 truncated polypeptide . transgenic animals are typically produced by introduction of a transgene by microinjection into pronuclei of one - cell embryos or a targeting vector into the host by electroporation , lipofection , or viral transfection of embryonic stem ( es ) cells . the transgenic animals that express the p25 polypeptide are suitable for use as models of disease and screening of potential therapeutic compounds including small molecules , proteins and polynucleotides . the invention also provides nonhuman or human cell lines to be derived by transformation of established cell lines or primary cell lines established directly from the nonhuman transgenic animal , for example neurons . it is obvious that the transgenic nonhuman animal can have additional genetic modifications by transgenic art or through traditional matings with other transgenic or naturally occurring animals to produce novel animals that serve as alternative disease models , drug screens or other applications . this includes the inactivation of the murine endogenous p35 gene through gene targeting in es cells and resultant murine p35 - deficient mice that becomes the preferred host for expression of the human p25 heterologous polypeptide . such heterologous transgenes may be integrated in nonhomologous chromosomal locations in the transgenic animal , typically derived by pronuclear injection or may be integrated by gene targeting in es cells by methods to inactivate the murine p35 gene and add linked sequences to direct expression of the heterologous human p25 sequences . in general , the invention encompasses methods for the generation and characterization of transgenic animals that express the human p25 polypeptide , a proteolytic fragment of p35 , both of which are an allosteric activators of cyclin dependent kinase 5 ( cdk5 ). the transgenic animals express this human protein in the presence of the endogenous homologue . the techniques and procedures are performed according to established protocols that are generally considered to be routine methods in the art and references are provided within this specification . the protein kinase cdk5 when associated with an allosteric activator , e . g ., p35 or p25 is reported in the literature to phosphorylate tau in vitro and in whole cells ( see background of the invention ). the transgenic mice thus produced establish the role of the human p25 in the formation of hyperphosphorylated tau in neurodegenerative conditions including alzheimer &# 39 ; s disease , parkinson &# 39 ; s disease , amyelolateral sclerosis , huntington &# 39 ; s chorea , stroke , traumatic brain injury , pick &# 39 ; s disease , neuraxonal dystrophy , multiple sclerosis , motor neuron disease , and spinocerebellar degeneration and other neurodegenerative diseases . it is apparent that the preparation of other transgenic animals that express the human p25 protein is easily accomplished including rats , hamsters , guinea pigs and rabbits . the transgenic animals that express the human p25 protein can be monitored for the level of expression and tau phosphorylation . it will be appreciated that under different conditions the level of expression and degree of tau phosphorylation will be inhibited in such an animal model . in particular , the screening of therapeutic agents that inhibit the activity of the human p25 protein will be greatly facilitated in animal models . it is apparent that the development of cell lines from the affected transgenic animals , ( e . g ., neurons ) will improve the throughput in which biochemical and pharmacological analysis of the human p25 protein can be assessed . particularly preferred animal models for p25 overexpression are transgenic anmals which express p25 as described above . such transgenic animals , particularly transgenic mice according to this invention , produce high quantities of p25 and hyperphosphorylated tau and neuronal death which may be detected according to the methods of the present invention . in accordance to this invention particular , the overexpression of p25 will be equal to or greater than endogenous p25 expression in such animals . further , this level of p25 overexpression results in tau hyperphosphorylation which is minimal in wild type animals . with such elevated levels of p25 , monitoring of hyperphosphorylation of tau and neuronal death is greatly facilitated . in particular , screening for compounds and other therapies for inhibiting tau phosphorylation are greatly simplified in animals overexpressing p25 according to this invention . agents are administered to test animals , such as test mice , which are transgenic and which overexpress p25 . particular techniques for producing transgenic mice which overexpress p25 are described below . it will be appreciated that the preparation of other transgenic animals overexpressing p25 may easily be accomplished , including rats , hamsters , guinea pigs , rabbits , and the like . in light of this disclosure , the effect of test compounds on the hyperphosphorylation of tau in the test animals may be measured in various specimens from the test animals . the effect of test agents on hyperphosphorylation of tau may be measured in various specimens from the test animals . in all cases , it will be necessary to obtain a control value which is characteristic of the level of tau phosphorylation and neuronal death in the test animal in the absence of the test compound ( s ). in cases where the animal is sacrificed , it will be necessary to base such control values on an average or a typical value from other test animals which have been transgenically modified to overexpress p25 but which have not received the administration of any test compounds or any other substances expected to affect the level of tau phosphorylation or neuronal death . once such control level is determined , test compounds can be administered to additonal test animals , where deviation from the average control value indicates that the test compound had an effect on the tau phosphorylation or neuronal death in the animal . test substances considered positive , i . e ., likely to be beneficial in the treament of ad or other neurodegenerative diseases , will be those which are able to reduce the level of tau phosphorylation or neuronal death preferably by at least 20 % and most preferably by 80 %. in addition there may be paired helical or straight filament formation in transgenic animals which overexpress p25 and display tau hyperphosphorylation . in these cases , test compounds can be administered to test animals and the reduction of filament formation monitored as a result of exposure to the compound . in addition , there may be behavioral alterations in the transgenic animals which overexpress p25 and display tau hyperphosphorylation . in these cases it will be necessary to obtain a control value from live animals performing a behavioral task ( e . g ., the measurement of locomotor activity ) in the test animal in the absence of test compound ( s ). such a control will also be determined in non - transgenic , wild type mice . the difference between the wild type and transgenic mice will serve as the outcome measure for the effects of compounds . once such control levels are determined , test compounds can be administered to additional test animals , where reduction in or reversal of the difference between the wild type and transgenic mice indicates that test compound has an effect on the behavioral test being measured . test substances considered positive , i . e ., likely to be beneficial in the treament of ad or other neurodegenerative diseases , preferably will be those which are able to reverse or , substantially reverse , or favorably modify the behavioral abnormality in the transgenic animal to the level found in wild type mice . test agents will be defined as any small molecule , protein , polysaccharides , deoxy or ribomucleotides , or any combination thereof that when added to the cell culture or animal will not adversely interfere with the cell or animal viability . agents that alter the level of human p25 expression and tau phosphorylation will be considered as candidates for further evaluation as potential therapeutics . the test compound will typically be administered to transgenic animals at a dosage of from 1 ng / kg to 100 mg / kg , usually from 10 ug / kg to 32 mg / kg . test compounds which are able to inhibit phosphorylation of tau are considered as candidates for further determinations of the ability to block tau phosphorylation in animals and humans . inhibition of tau phosphorylation indicates that cdk5 / p25 activity has been at least partly blocked , reducing the amount of cdk5 / p25 available to phosphorylate tau . the present invention further comprises pharmaceutical compositions incorporating a compound selected by the above - described method and including a pharmaceutically acceptable carrier . such pharmaceutical compositions should contain a therapeutic or prophylactic amount of at least one compound identified by the method of the present invention . the pharmaceutically acceptable carrier can be any compatible , non - toxic substance suitable to deliver the compound or compounds to an intended host . sterile water , alcohol , fats , waxes and inert solids may be used as the carrier . pharmaceutically acceptable adjuvants , buffering agents , dispersing agents , and the like may also be incorporated into the pharmaceutical compositions . preparation of pharmaceutical compositions incorporating active agents is well described in the medical and scientific literature . see , for example , remington &# 39 ; s pharmaceutical sciences , mack publishing company , easton , pa ., 16 th ed ., 1982 , the disclosure of which is incorporated herein by reference . the pharmaceutical compositions just described are suitable for systemic administration to the host , including both parenteral , topical , and oral administration . the pharmaceutical compositions may be administered parenterally , i . e ., subcutaneously , intramuscularly or intravenously . thus , the present invention provides compositions for administration to a host where the compositions comprise a pharmaceutically acceptable solution of the identified compound in an acceptable carrier , as described above . non - human animals comprising transgenes which encode p25 can be used commercially to screen for agents having the effect of preventing or reducing the phosphorylation of tau and neuronal death . such agents can be developed as pharmaceuticals for treating hyperphosphorylation of tau and ad amongst other neurodegenerative diseases . for example , the p53 knockout mice of donehower et al . ( 54 ) have found wide acceptance as commercial prouducts for carcinogen screening and the like . the transgenic animals of the present invention exhibit abnormal tau phosphorylation and can be used for pharmaceutical screening and as disease models for neurodegenerative diseases and cdk5 / p25 and tau biochemistry . such animals have many uses including but not limited to identifying compounds that affect tau hyperphosphorylation ; in one variation , the agents are thereby identified as candidate pharmaceutical agents . the transgenic animals can also be used to develop agents that modulate cdk5 or p25 expression and or stability ; such agents can serve as therapeutic agents to treat neurodenerative diseases . the p25 overexpressing mice of the invention can also serve as disease models for investigating tau - related pathologies ( e . g ., ad , pick &# 39 ; s disease , parkinson &# 39 ; s disease , frontal temporal lobe dementia associated with chromosome 17 , stroke , traumatic brain injury , mild cognitive impairment and the like ). such transgenic animals can be commercially marketed to researchers , among other uses . having described the invention in general terms , reference is now made to specific examples . it is to be understood that these examples are not meant to limit the present invention , the scope of which is to be determined by the appended claims . the plasmid pnse - p25 ( rat neuron specific enolase promoter , human p25 transgene ) contains dna from 4 different sources : the rat neuron specific enolase promoter ; the human cdna for the p25 catalytic fragment of the p35 protein ; the sv40 polyadenylation signal sequence and a commercially available plasmid vector . the transgene is isolated by bamh1 restriction enzyme digestion of pnse - p25 . this restriction digest releases the nse - p25 transgene ( 3212 bp ) from the plasmid cloning vector psp72 ( 2462 bp ) ( promega , madison , wis .). the rat neuron specific enolase ( nse ) promoter sequence is 1826 bp ( seq id no : 1 ) and has been demonstrated to efficiently express dna coding sequences in neurons of transgenic mice ( 55 ). the coding region for the human p25 sequence is 633 bp . ( seq id no : 2 ). the 3 - prime untranslated and sv40 polyadenylation sequence are 688 bp ( seq id no : 3 ). the microinjected nse - p25 transgene is 3212 bp ( seq id no : 4 ). enzymatic reactions of recombinant dna , including ligations , restriction , endonuclease digestions , dna synthesis reactions , single strand fill - in reactions , and bacterial transformations performed are well established procedures as described by sambrook , j ., fritsch , e . f . and maniatis , t . molecular cloning : a laboratory manual . 2nd edition , cold spring harbor press , new york , 1989 . the sv 40 polyadenylation sequence ( seq id no : 3 ) was cloned into the commercially available plasmid psp72 ( seq id no : 5 ) at xba1 and bstx1 sites . this plasmid , designated psp - svpa , served as the backbone vector for the construction of the nse - p25 transgene plasmid . the cdna for the human p25 catalytic fragment was derived using polymerase chain reaction ( pcr ). the human p35 full length cdna was used as the template for amplification of the p25 insert . we received the p35 template dna from dr . david auperin of pfizer ; the human p35 sequence was cloned into the ecor1 site of the pfastbac1 , baculoviral expression vector ( gibco - brl , gaithersburg , md .). the pcr primers were designed to have not1 restriction sites outside the coding sequence to facilitate transgene construction . the forward primer also introduced a new atg start codon adjacent to the gcc ( ala codon at position 99 of the human p35 sequence ). the primers ( submitted in 5 - prime to 3 - prime orientation ) were p25 - forward : gcggccgcatggcccagcccccaccggcccagccgcctgca ( seq id no : 6 ) and p25 - reverse : ctcctcctaggcctggaatcgg tga ggcggccgc ( seq id no : 7 ). the atg is introduced start codon , the tga is the stop codon and the gcggccgc are the not1 restriction sites added to the pcr primers . the resulting pcr product is 648 bp containing the entire 633 bp human p25 cdna sequence with engineered atg start site ( seq id no : 2 ) and an additional 15 bp from addition of the not 1 cloning sites . the 648 bp fragment was subcloned into the pcr2 . 1 ( invitrogen , carlsbad , calif .) vector for verification of sequence prior to use as insert in the final transgene . the confirmed 648 bp not 1 fragment was ligated into the not 1 restriction site of psp - svpa producing the plasmid psp - svpa : p25 . the rat neuron specific enolase ( nse ) promoter was isolated from the plasmid pnse - lacz obtained from dr . j . g . sutcliffe of the research institute of scripps clinic ( lajolla , calif .). the rat nse promoter sequence ( seq id no : 1 ) was isolated by ecor1 and hind iii restriction digestion of the pnse - lacz plasmid . the 1 . 8 kb fragment was extracted by agarose gel electrophoresis and purified . the 5 - prime single strand overhangs generated by the restriction enzymes were filled by klenow reaction to produce the rat nse promoter fragment with double stranded blunt ends . the 1826 bp rat nse blunt end promoter fragment was blunt end ligated into the sma 1 restriction site of psp - svpa : p25 . the correct orientation of the rat nse promoter within the psp - svpa : p25 was verified by double restriction digests of bamh1 and xho1 . plasmids with the correct orientation were assigned the designation as pnse - p25 . the entire sequence of the 3212 bp nse - p25 transgene ( seq id no : 4 ) was analyzed and verified by automated sequencing on abi 373 sequencer ( foster city , calif .) using the standard dye - terminator chemistry protocol outlined by abi . all plasmids were grown in dh5alpha bacterial cells ( gibco brl gaithersburg md .). the 3212 bp nse - p25 transgene dna fragment was excised from the pnse - p25 plasmid by bamh1 restriction endonuclease reaction . the 3 . 2 kbp fragment was isolated by electroelution ( 50v , 3 hrs ) after electrophoresis in 1 % agarose gel ( fmc bioproducts , rockland me .). the fragment was further purified by on a schleicher and schuell ( keene , n . h .) elutip - d column following protocols established by the manufacturer for dna purification prior to murine embryo microinjection . production of transgenic mice by pronuclear microinjection was carried out by published procedures as outlined in hogan , b . et al manipulating the mouse embryo : a laboratory manual 2nd edition , cold spring harbor laboratories , new york , 1994 . pronuclear stage embryos from f1 females mice of the strain fvb / n ( charles river labs , wilmington , mass .) were obtained after superovulation with 5 international units ( iu ) of follicle stimulating hormone from pregnant mare serum ( sigma st louis , mo .) and 2 . 5 i . u . human chorionic gonadotropin ( sigma ). the actual microinjection procedure was performed as described by wagner , t . et al . ( 56 ) except that the embryos were transferred immediately to pseudopregnant cd - 1 recipient females ( charles river laboratories , wilmington , mass .) for development of embryos to term . mice resulting from the reimplantation events were tested for the presence of the nse - p25 transgene by pcr analysis of genomic dna isolated from tail biopsies at 3 weeks of age . the mice that demonstrated positive for the presence of the nse - p25 transgene were mated with wild type fvb / n mice ( charles river laboratories , wilmington , mass .) of the opposite sex . offspring of these matings were tested for germline transmission by pcr analysis or southern blot analysis of genomic dna isolated from tail biopsies at 3 weeks of age . transgenic lines were produced from 7 founder transgenic mice and were maintained by breeding to wild type fvb / n mice and pcr genotyping for the presence of the nse - p25 transgene . the experiments described below were done in mice heterozygous for the inserted transgene . these mice were derived by breeding mice positive for the transgene insert with fvb / n wild - type animals and screening offspring by pcr for presence of the nse - p25 transgene sequences . whole brains from 1 or 4 month old transgenic ( tg ) and wild type ( wt ) mice were removed and snap frozen in liquid nitrogen . amygdala , thalamus and cortex were dissected and homogenized in 1 ml of lysis buffer ( as described in 37 ). the samples were boiled for 10 minutes and then centrifuged at 13 , 000 rpm . protein concentrations of the resulting supernatants were determined by the pierce bca method ( bca micro protein assay , catalog # 23225 , pierce , rockford ill .). ten micrograms of each sample were electrophoresed on an sds polyacrylamide gel ( 57 ) and transferred to problott protein paper for western blot analysis [ western blotting protocols , ecl detection , amersham life science arlington heights , ill . ( 1995 )]. non specific sites were blocked by incubating the blots in 5 % nonfat milk in tris - buffered saline ( 20 mm tris base , 127 mm nacl , 3 . 8 mm hcl ph 7 . 6 , sigma ) included 0 . 1 % tween - 20 ( tbs - t ). primary antibodies were diluted in 5 % nonfat milk \ tbs - t . the blots were placed in the diluted antibody solutions and rotated for 1 hr at 23 ° c . the western blots were then washed for 30 min in tbs - t . secondary horseradish peroxidase linked antibodies were diluted in 5 % nonfat milk \ tbs - t . the blots were incubated with the secondary antibody solution and rotated for 45 min at 23 ° c . the blots were then washed for 30 min in tbs - t . equal volumes of amersham &# 39 ; s ecl developing solutions a and b were mixed together . the western blots were incubated for 1 min in the developing solution . the blots were wrapped in saran ® wrap and then exposed to imaging film ( kodak rochester n . y . x - omat ar , catalog # 165 1454 ). western blots of the amygdala ( 1 ), thalamus ( 2 ) and cortex ( 3 ) from 3 mice were probed with an antibody specific for p25 and p35 / 39 ( gift of l .- h . tsai , harvard medical school , fig1 a ). while detection of p35 / 39 is apparent , no p25 is detected in wild type mice . when the same analysis is performed in the transgenic mice ( fig1 b ), robust expression of p25 in addition to the constitutive expression of p35 is detected . these results confirm that expression of the transgene is robust in the p25 transgenic animals . tissues collected from scheduled sacrifice mice were perfusion - fixed in situ with 10 % neutral - buffered formalin or 4 % paraformaldehyde , whereas tissues collected from mice found dead were immersion fixed in formalin . following fixation , trimmed tissues were dehydrated through graded alcohols and embedded in paraffin . standard cross sections of brain were identified by analogy to figures in the mouse brain in stereotaxic coordinates ( franklin and paxinos , 1997 ). paraffin sections ( 8 μm thick ) were stained with modified bielschowsky silver stain and , immunohistochemically , with an antibody directed against phosphorylated neurofilament ( smi 34 , commercially available from sternberger monoclonals , inc lutherville , md .). for immunohistochemical studies , transgenic and wild type mice were deeply anesthetized with sodium pentobarbital ( 60 mg / kg , i . p .) and perfused through the ascending aorta with 20 ml of 0 . 1m napo 4 containing 0 . 9 % nacl ( pbs , 7 . 4 ) followed by 30 ml of fixative containing 4 % paraformaldehyde in 0 . 1m napo 4 ( pb , 7 . 4 ) buffer . the brains were removed , cryoprotected for 48 hr in pbs containing 20 % sucrose , frozen in a bed of pulverized dry ice , and then cut into 35 micron sections on a sliding microtome . consecutive 1 in 18 series of sections were collected in 0 . 1m pb and processed for immunohistochemistry as described below . for immunohistochemistry , sections were incubated for 1 hr in 50 mm tris buffer , ph 7 . 4 containing 0 . 9 % nacl ( tbs ) and 0 . 1 % triton x - 100 . the sections were then washed in tbs and incubated for 1 hr in antibody vehicle ( 4 % goat or horse serum , 0 . 1 % triton x - 100 in tbs ). after additional washes in tbs , sections were incubated overnight at 4 ° c . in vehicle containing the primary antibody . following overnight incubation in the primary antibody , sections were washed in tbs , incubated for 1 hr in vehicle containing biotinylated horse anti - mouse or goat anti - rabbit antibody ( as per manufacturer , vector labs , burlingame , calif . ), washed in tbs , and then incubated for 1 hr in tbs containing abc / horseradish peroxidase reagent ( vector labs , burlingame , calif .). after additional washes in tbs , immunolocalization products were visualized by developing sections for 3 - 5 min in 50 mm tris buffer ( ph 7 . 6 ) containing 0 . 04 % diaminobenzdine ( dab ) and 0 . 003 % h 2 o 2 . sections were subsequently washed in the tris buffer , mounted onto slides and dehydrated and coverslipped using dpx ( fluka , ronkokoma , n . y .). used at 1 : 1 , 000 ( 200 ng / ml ), mouse monoclonal which recognizes ser 202 and thr 205 of human phosphorylated tau . ( innogenetics , inc . zwij + ndrecht ( belgium )) used at 1 : 20 , 000 , mouse monoclonal which recognizes ser 396 and thr 404 of human phosphorylated tau . ( gift from v . lee , university of pennsylvania ) used at 1 : 7 , 000 , rabbit polyclonal which recognizes total tau . ( accurate chemical and scientific corp westbury , n . y .). used at 1 : 1000 , mouse monoclonal which recognizes phosphorylated neurofilament . h ( sternberger monoclonals , inc ., lutherville md .) brains from four month old transgenic and wild type mice were compared for the presence of tau and neurofilament phosphoepitopes by immunohistochemistry . at - 8 and phf - 13 , monoclonal antibodies which recognize phosphorylated ser - 202 and thr - 205 , and ser - 396 , 404 , respectively , labeled neurons in amygdala ( fig2 and 4 , respectively ), thalamus / hypothalamus and cortex adjacent to external capsule ( not shown ) of transgenic animals but not in corresponding regions of wild - type animals ( fig3 and 5 ). in addition , staining for total tau revealed numerous cell bodies in p25 transgenic animals ( fig6 ) while mostly axons and very few cell bodies were labeled in wild type mice ( fig7 ). a monoclonal antibody ( smi34 ) recognizing phosphorylated neurofilament h also showed increased immunostaining in these three brain regions and spinal cord of transgenic mice ( amygdala is depicted in fig8 ), but not in wild type ( fig9 ). all three of these markers are known to be increased in alzheimer brain relative to age - matched control . in alzheimer &# 39 ; s brain , pathological changes in neurons containing altered tau proteins have classically been identified using silver - based staining methods . we found that , in the transgenic mice of this invention , neurons in the same three brain regions described above and spinal cord show positive labelling using the modified bielschowsky silver stain ( amygdala , fig1 ). this staining was once again absent in wild - type control animals ( fig1 ). we also detected increases in silver staining and phosphorylated neurofilament h in obviously enlarged axons in the spinal cord of transgenic mice ( fig1 and 13 , respectively ), while in wild type mice , axons were of the expected diameter and neurofilament staining was normal ( fig1 ). with regard to at - 8 immunoreactivity , most labelled neurons in the amygdala exhibited well - defined , densely staining soma , often with a swollen , dystrophic hillock and contorted axon ( fig2 ). less commonly , diffuse labelling of unidentified cells was seen , and occasionally cells having astroglial or microglial morphology were labelled . in cortex adjacent to external capsule , the majority of at - 8 staining was found in intensely - labelled neuronal cell bodies , often accompanied by axons when present in the plane of the section . thalamic / hypothalamic staining was more diverse , including both neuronal staining as seen in other sections . histologic examination of brain and spinal cord from p25 tg mice revealed changes in neurons and axons of brain or spinal cord ( n = 19 ) but not in these tissues from 10 age - matched , wild - type controls . axonal changes were most pronounced in the spinal cord ( cervical and thoracic ) and consisted of marked dilation of axoplasm to diameters of 20 to 50 μm . dilated axons were filled with both silver staining and phosphorylated neurofilament h ( fig1 , 13 ). a given cross section of affected cord generally contained between 10 and 100 overtly dilated axons distributed among all funiculi of white matter . luxol fast blue staining illustrated dissolution of the internal portion of myelin sheath of affected axoris ( not shown ). 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