in patients with alzheimer 's disease ( ad ) , plaques 
 comprised of aggregated -amyloid peptides ( a ) accumulate in specific areas of the brain as a 
 consequence of the proteolytic processing of the single - pass 
 transmembrane amyloid precursor protein ( app )   . 

these a deposits trigger prolonged inflammation , neuronal 
 death , and progressive cognitive decline . 

a peptides are derived from app by -site cleavage by an aspartic protease ( bace ) producing a membrane - bound 
 cooh - terminal c99 fragment followed by a complex 
 proteolytic event ( involving presenilin and nicastrin ) at the c99 
 transmembrane - localized  position [ 35 ] .
an 
 alternative app processing pathway also exists in which 
 membrane - proximal ( -site ) cleavage by matrix metalloproteinases ( tace , adam 10 ) replaces  position utilization producing a membrane - anchored c83 fragment . 
 subsequent -site processing of the c83 product results in generation 
 of the nontoxic p3 peptide   [ 3 , 
 6 ] .
the broad - spectrum protease plasmin also degrades a [ 79 ] 
 and activation of plasmin decreases a peptide levels   . 

plasmin - mediated proteolysis of app , moreover , appears to involve the  
 site ( either as a direct or indirect target ) resulting in decreased 
 a production , thus suggesting a protective role for the plasmin cascade in the 
 central nervous system .
indeed , plasmin levels in the brains of ad 
 patients are considerably reduced  
 further supporting a causal relationship between deficient activity of the 
 plasmin - generating proteolytic system and accumulation of a in the progression of ad .
several members of the serine protease inhibitor ( serpin ) 
 superfamily exhibit neurotrophic , neuroprotective , or 
 neuropathophysiologic activities depending on the specific cell 
 type and pathways involved   . 

these include serpinf1 , serpini1 ( neuroserpin ) , serpine1 ( plasminogen activator 
 inhibitor type-1 ; pai-1 ) , serpine2 ( nexin-1 ) , and serpina3  
 . 

pai-1 , in particular , has multifunctional roles in the central 
 nervous system as it both maintains neuronal cellular structure 
 and initiates signaling through the erk pathway 
 . 

pai-1 directly influences the plasmin - dependent pericellular 
 proteolytic cascade by regulating the conversion of plasminogen to 
 plasmin by urokinase- and tissue - type plasminogen activators 
 ( upa / tpa ) ( figure 1 ) .
pai-1 immunoreactivity in the central nervous system of ad 
 patients was associated with senile plaques and ghost tangle 
 structures   consistent with the earlier 
 colocalization of pa and pai-1 in plaque structures 
  which are sites of 
 sustained inflammation .
recent findings 
 in tg2576 and tgcrn8 transgenic mice , engineered to express brain - targeted 
 swedish mutant a and the double swedish / v717f mutant 
 a , respectively , under control of the hamster prion 
 promoter and exhibit age - dependent a plaque development ( at 12 and 3 months , resp ) as well as cognitive 
 deficiencies , established that tpa activity 
 was significantly decreased compared to controls 
 .
this decline 
 correlated with corresponding increases in pai-1 expression specifically in 
 areas of the brain where tpa activity was reduced ( hippocampus , 
 amygdala ) . since direct a peptide injection increased 
 pai-1 expression and whereas a removal from the 
 hippocampal region required both tpa and plasminogen , it appears 
 that a functional tpa - plasmin axis is required for a clearance .  
while pai-1 may be 
 neuroprotective in specific acute injury settings ( eg , tpa - triggered neuronal 
 apoptosis ) , chronically elevated pai-1 
 levels likely promote a accumulation by inhibiting plasmin - dependent 
 degradation ( figure 1 ) .
several reports described elevated tgf-1 levels in brain 
 biopsies from patients with parkinson 's disease , ad , and stroke 
 [ 2022 ] .
this growth factor is likely to influence the 
 onset and progression of ad at several levels .
increased brain 
 expression of tgf-1 correlates with a angiopathy , 
 and transgenic mice that overexpress tgf-1 in astrocytes 
 exhibit early onset a deposition . 

tgf-1 , moreover , induces astrocyte app expression through a 
 tgf-1-responsive agac smad - binding element in the app 
 promoter ; subsequent a production , moreover , was enhanced 
 by tgf-1 signaling . 

since the pai-1 gene is also transcriptionally upregulated by tgf-1 [ 19 , 
 25 ] , 
 the coordinate overexpression of pai-1 and increased a generation in response to elevated tgf-1 in the brains of 
 ad patients may dispose to disease progression   . 

collectively , these findings raise the possibility that targeting 
 tgf-1-inducible genes ( eg , pai-1 , app ) may have 
 therapeutic benefit in the setting of ad .
the regulation of tgf-1-activated genes ( ie , pai-1 ) is 
 largely transcriptional [ 19 , 
 25 , 27 , 
 28 ] with the pai-1 gene 
 subject to complex combinatorial expression controls involving the 
 major transcription effectors p53 , sp1 , and members of the myc 
 family   [ 19 , 
 29 , 
 30 ] .
prominent tgf--response elements 
 in the human pai-1 promoter include the hexanucleotide e box motif 
 ( 5-cacgtg-3 ; as in the pe1 , pe2 , hre-2 sites ) and closely 
 related sequences recognized by the basic helix - loop - helix / leucine 
 zipper ( bhlh - lz ) transcription factors of the myc family ( eg , myc , 
 max , tfe3 , usf-1 , and usf-2 ) 
 [ 3136 ] . 

this e box element likely functions , 
 therefore , as a  platform  for recruitment of both positive and 
 negative regulators of pai-1 expression [ 3739 ] . 

recent uv crosslinking and tethered dna affinity chromatographic analyses 
 identified the bhlh - lz protein upstream stimulatory factor-1 
 ( usf-1 ) as a major pai-1 e box - recognition factor . 

specific e box mutations that ablate usf-1 binding to a pai-1 
 target deoxyoligonucleotide probe ( ca  tc ) effectively attenuated tgf-1-stimulated pai-1 promoter - driven cat reporter activity 
 . the human pai-1 promoter , however , 
 harbors several additional 
 tgf--responsive elements , including three e 
 box - adjoining smad sequences located 
 just 5 of the pe2 site [ 32 , 
 33 , 35 , 
 41 ] .
since an engineered 
 two - base - pair mutation cacgtg  caattg 
 in a serum - responsive pai-1 e box attenuated growth state as well 
 as tgf-1-dependent transcription , 
 this same dinucleotide substitution was incorporated into a 
 luciferase reporter construct bearing the immediate 806 base pairs 
 of the human pai-1 5 upstream region .
initial truncation 
 approaches did , in fact , confirm that a major ( albeit not the 
 only ) tgf-1-responsive element resided within the most 
 proximal 606 base pairs of the human pai-1 promoter 
 . 

specific disruption of the pe2 region e box element by 
 site - directed mutagenesis significantly attenuated 
 tgf-1-mediated pai-1 transcription 
 ( figure 2 ) . 

the consequences of usf binding 
 to the pai-1 pe2 e box site may be more complex , however , than 
 simple motif occupancy .
indeed , certain e box - recognition factors 
 including usf-1 and usf-2 effectively induce dna bending . 

cooperative interactions between sp1 and/or p53 with usf proteins , 
 for example , may be dependent on usf - initiated modifications to 
 dna conformation allowing distally spaced factors important in 
 expression control to interact with resulting effects on pai-1 
 transcription .
chromatin immunoprecipitation recently confirmed that the pai-1 
 pe2 e box site is an in vivo usf target motif  
 . 

since an intact consensus pe2 region e box sequence is necessary 
 for a maximal transcriptional response to growth factors 
 , it was important to identify any additional 
 requirements for pe2 e box - occupancy that might influence site 
 residence including the smad - binding agac elements implicated in 
 tgf-1-dependent app expression . 

pe1 and pe2 probes recognition appeared dependent solely on an 
 intact 5-cacgtg-3 motif since nuclear factor binding to individual pe1 and pe2 target 
 constructs was successfully blocked by short double - stranded 
 deoxyoligonucleotides containing a consensus e box flanked by 
 non - pai-1 sequences whereas a mutant e box ( 5-caattg-3 ) 
  bait  failed to compete 
 . 

it was important , however , to confirm these results using site - specific mutants within the 
 context of native pai-1 promoter sequences ( eg , the pe2 region 
 backbone ) in order to assess the potential contributions of the 
 smad - binding elements , e box flanking nucleotides ( such as the aat 
 trinucleotide  spacer  between the pe2 e box and the first 
 upstream smad site ) , and the cacgtg motif to nuclear protein 
 binding ( figure 2 ) .
a recent study established that 
 the major protein / dna interactions in the pe2 segment were , in 
 fact , e box - dependent and did not require accessory sites since 
 mutation of all three smad - binding sites ( agac  cttg ) or removal of the att spacer did not affect usf 
 occupancy of the pe2 region e box . 
 while the cacgtg  core  is a target for occupancy by at least seven 
 members of the bhlh - lz transcription factor family ( usf-1 , usf-2 , c - myc , max , 
 tfe3 , tfeb , tfii - i ) , usf proteins have a preference for c or t at 
 the 4 position in the presence of mgcl2 
 . 
 indeed , the human pai-1 gene has a t at the 4 site of the pe2 
 region e box as well as a purine at + 4 and 5 and a pyrimidine at + 5 
 ( a5t4c3a2c1g+1t+2g+3g+4c+5 ) , all of which facilitate usf binding 
 .
chromatin immunoprecipitation 
 of the pe2 region e box site in the human pai-1 gene , moreover , indicated a dynamic 
 occupancy by usf subtypes ( usf-1 versus usf-2 ) as a function of 
 growth state .
an exchange of pe2 e box usf-1 
 homodimers with usf-2 homo- or usf-1/usf-2 heterodimers , 
 furthermore , closely correlated with pai-1 gene activation .
this 
 switch may well determine the transcriptional status of the pai-1 
 gene in quiescent versus growth factor - stimulated culture 
 conditions [ 38 , 
 45 ] .
site occupancy and transcriptional 
 activity additionally require conservation of the pe2 core e box 
 structure as the cacgtg  cacgga and tccgtg dinucleotide substitutions ( in the rat gene ) 
  and 
 a cacgtg  caattg or tccgtg replacement 
 ( in the human gene ) , with retention of pai-1 flanking sequences , 
 resulted in loss of both competitive binding and growth 
 factor - dependent reporter activity [ 19 , 
 44 ] .
the 
 cacgtg  tccgtg mutation is particularly 
 relevant since bhlh proteins with e box - recognition activity have 
 a conserved glutamate important for interaction with the first two 
 nucleotides ( ca ) in the e box motif
. 
 these data are also consistent with the known hexanucleotide preference ( cacgtg or 
 cacatg ) of usf proteins [ 39 , 
 47 , 48 ] . 
 to further dissect the role of usf in tgf1-mediated pai-1 transcription 
 in vivo , a dominant - negative usf construct ( a - usf )
a - usf effectively titers away functional usf proteins by forming highly 
 stable interactions with native usf proteins ; such usf / a - usf 
 heterodimers , however , are unable to bind dna due to replacement 
 ( in the a - usf construct ) of the basic dna - binding residues with an 
 acidic domain .
a - usf transfection effectively 
 attenuated tgf-1-induced pai-1 expression establishing the 
 importance of usf family transcription factors in pai-1 gene 
 control [ 19 , 
 50 , 51 ] .
the molecular mechanisms associated with the tgf-1-initiated e box - dependent pai-1 gene control and 
 the collateral smad - mediated app induction / tgf--directed a processing in specific central nervous system 
 cell types remain to be clarified .
the available data , however , 
 clearly suggest that these two responses to tgf-1 are linked in the pathophysiology of human neurodegenerative disease . 

it has become apparent , moreover , that pai-1 overexpression is a 
 likely major contributory if not a causative event in ad 
 progression .
our current understanding of the pathways utilized by 
 the tgf-1 to stimulate the pai-1 transcription 
 ( summarized in figure 3 ) indicate that this 
 growth factor activates a kinase cascade , at least partially as a 
 function of epidermal growth factor receptor mobilization ( either 
 through the release of the appropriate ligands or the direct 
 receptor transactivation ) , involving mek , erk1/2 , and perhaps p38 
 [ 4951 ] .
pharmacological approaches , use of 
 dominant - negative constructs , and kinase assays suggest that 
 src family kinases and ras gtpase are 
 upstream of mek - erk - p38 in this model of induced pai-1 expression 
 [ 50 , 51 ] . 

map kinases , in turn , interact with nuclear 
 transcription factors including members of the usf family that , 
 once phosphorylated , bind as dimers to e box motifs in the pai-1 
 promoter to modulate gene expression [ 19 , 
 26 , 38 , 
 40 , 50 , 
 51 ]
. 
 genetic perturbation of pai-1 synthesis in specific areas of the 
 brain ( with dominant - negative usf or pai-1 antisense vectors ) or 
 delivery of pai-1 neutralizing antibodies may effectively 
 stimulate upa- and/or plasmin - dependent target substrate 
 degradation ( eg , a ) or at least attenuate the rate of 
 a accumulation ( figures 1 and 
 3 ) .
the continued identification of regulatory points in the pai-1 
 expression control network ( figure 3 ) and recent 
 identification of tgf-1-response sites ( as well as the 
 involved nuclear factors ) in the app and pai-1 promoters 
 [ 19 , 38 , 
 40 , 52 ] 
 may provide new molecular targets for the 
 therapy of neurodegenerative syndromes associated with pai-1 
 upregulation .
indeed , specific serpins ( including pai-1 ) have 
 already been suggested as potential novel therapeutic targets for 
 stroke and cerebral ischemia . 
 the tgf-1 gene , furthermore , is also usf - regulated 
 
 suggesting that interference with usf - dependent transcriptional events may have 
 widespread therapeutic implications .
tpa and upa convert 
 plasminogen to the active , broad - spectrum , protease plasmin both 
 at the cell surface and in the immediate pericellular space . 

plasmin , in turn , degrades target substrates ( eg , app , 
 a ) directly as well as indirectly through downstream activation of 
 matrix metalloproteinases ( mmps ) . inhibition of mmp activity 
 ( gm6001 , timp ) has confirmed their participation in 
 plasmin - initiated proteolysis .
most importantly , this cascade is 
 effectively attenuated by overexpression ( or exogenous addition ) 
 or pai-1 which blocks tpa and upa catalysis inhibiting , thereby , 
 plasmin generation .
topography of the pai-1p806-luc reporter construct illustrating the 
	            two ( pe1 and pe2 ) e box sequences .
site - directed mutagenesis and luciferase 
 assays clearly indicated that an intact e box at the pe2 site is required for maximal 
 tgf-1-induced pai-1 transcription in human epithelial cells 
 .
pathways involved in 
 the regulation of pai-1 expression and function in response to 
 tgf-1 stimulation .
positve influences are depicted in 
 black arrows ; effective inhibitors defined pharmacologically or by 
 use of dominant - negative constructs ( dn ) are highlighted in red 
 ( detailed in ) . 

 amyloid peptide ( a ) aggregates , derived from initial -site proteolytic processing of the amyloid precursor 
 protein ( app ) , accumulate in the brains of alzheimer 's disease 
 patients . 
 the plasmin - generating cascade appears to serve a 
 protective role in the central nervous system since 
 plasmin - mediated proteolysis of app utilizes the  site , eventually generating nontoxic peptides , and plasmin also degrades 
 a. the conversion of plasminogen to plasmin by tissue - type 
 plasminogen activator in the brain is negatively regulated by 
 plasminogen activator inhibitor type-1 ( pai-1 ) resulting in 
 attenuation of plasmin - dependent substrate degradation with 
 resultant accumulation of a. pai-1 and its major 
 physiological inducer tgf-1 , moreover , are increased in 
 models of alzheimer 's disease and have been implicated in the 
 etiology and progression of human neurodegenerative disorders . 
 
 this review highlights the potential role of pai-1 and tgf-1 in this process . 
 current molecular events associated with 
 tgf-1-induced pai-1 transcription are presented with 
 particular relevance to potential targeting of pai-1 gene 
 expression as a molecular approach to the therapy of 
 neurodegenerative diseases associated with increased pai-1 
 expression such as alzheimer 's disease . 
INTRODUCTION
PLASMIN-ACTIVATING SYSTEM IN ALZHEIMER'S  DISEASE
CONTROLS ON TGF-1 TARGET GENES: THE PAI-1 MODEL
SEQUENCE REQUIREMENTS FOR USF OCCUPANCY
OF THE PE2 REGION E BOX MOTIF
MAPPING THE TGF-1-INDUCED PAI-1 SIGNALING AND
TRANSCRIPTIONAL CONTROL NETWORKS: OPPORTUNITIES FOR THERAPEUTIC
INTERVENTION
Figures and Tables