Patent Application: US-53159595-A

Abstract:
the invention provides a method for regulating neuronal degeneration resulting from injury to nervous tissue . the method includes regulating the expression or activity of protease , preferably tissue - type plasminogen activator , by microglial cells . alternatively , the method involves regulating microglial activation in response to injurious conditions . the method , in an alternative embodiment , includes detecting tpa expression or activity , or detecting microglial activation . in addition , the method includes assessing the susceptibility of a human or animal subject to seizure , and may involve detecting the activity or expression of tpa , and may further involve comparing a measured level of tpa expression or activity with a reference level associated with a given probability of seizure .

Description:
neuronal cell death occurs during normal development of the nervous system as well as in various pathological conditions . the molecular mechanisms by which this degeneration occurs have previously been unknown . in addition , the role which tpa plays in neuronal plasticity has previously been unclear . using excitotoxins to produce neuronal structural changes , we have now unexpectedly found that tpa is required for neuronal degeneration in the hippocampus . additionally , we have found that , surprisingly , mice which are genetically deficient for tpa are less susceptible to pharmacologically - induced seizure than normal or wild - type mice . given these new findings , several implications are now evident regarding the neuronal degeneration associated with seizure , and methods by which either or both of these phenomena may be detected and / or inhibited . these results identify a functional role for tpa in neuronal degeneration and remodeling . it is now established , as described in the examples provided hereinbelow , that tpa is directly associated with the susceptibility for seizure as well as with the degenerative changes which accompany seizure , and presumably other insults to the nervous system . accordingly , methods are now available which exploit these relationships for both in vitro and in vivo intervention . specifically , the invention provides methods of regulating neuronal degeneration and / or seizure , as well as for determining the predisposition of individual human or animal subjects to seizure and neuronal degeneration . methods are known for the molecular genetic regulation of structural gene expression . any of the known methods may be adapted for use in the present invention for the regulation of tpa expression . for example , antisense rna is a technique which has shown broad applicability both in vitro and in vivo . specifically , antisense rna complementary to both coding and noncoding regions of tpa rna have been shown to selectively block tpa synthesis ( ref . 16 ). other methods of controlling tpa expression may also be employed consistent with the present invention . such methods include , for example , gene transfer techniques in which a gene encoding a less active form of tpa is introduced by virus or other vector into microglial cells ( ref . 17 ), and regulation of transport and / or release of newly synthesized tpa molecules . numerous such methods are described , for example , in meyers , r . a ., ed ., molecular biology and biotechnology , vch publishers , inc ., new york ( 1995 ) ( ref . 18 ), the entire disclosure of which is incorporated by reference herein . more generally , we have now found that microglial activation is directly associated with increase in tpa expression by those cells . it would appear , therefore , that inhibition of microglial activation is a suitable means of accomplishing the stated end of reducing tpa expression . inhibitors of microglial activation are known . for example , mk - 801 inhibits microglial activation and also inhibits tpa expression . mk - 801 is a non - competitive antagonist of the nmda receptor . presumably , other nmda receptor inhibitors would effectively inhibit tpa expression . alternatively , the method of the invention includes methods of regulating tpa activity once it has been expressed . for example , since tpa is a proteolytic enzyme , like other enzymes it may be inhibited . both exogenous and endogenous ( ref . 19 ) inhibitors of tpa are known , which inhibit tpa activity either on a temporary or a permanent basis . one such endogenous inhibitor , plasminogen - activator - inhibitor - 1 ( pai - 1 ) is recognized as a potent and specific inhibitor of tpa ( refs . 20 - 21 ). other methods of interfering with tpa activity are also known , including interfering with the interaction of tpa with cellular receptors and intercellular matrix components . small molecule inhibitors would also be suitable for this purpose . for inhibiting microglial activation and / or tpa activity or expression , the agents suitable for these effects preferably act substantially selectively , incurring few if any side effects . the mode of administration may be determined by the skilled artisan based on the physical and chemical properties of the active agent . enteral or parenteral administration is contemplated . parenteral administration routes include injection , e . g ., intravenous injection of the agent in composition with a suitable pharmacologically acceptable diluent together with other active and / or inactive ingredients , excipients , etc . for example , two or more agents active in accordance with the invention may be administered together , thereby achieving additive or even synergistic effects . typically , inasmuch as the site of action of the agent is desirably in the central nervous system , more preferably the brain , the agent should be capable of reaching the nerve tissue without being substantially impeded by the blood - brain barrier . this permits administration at a site in the body which is convenient and relatively non - invasive . otherwise , if the agent is not substantially resistant to metabolic degradation or is incapable of traversing the blood brain barrier , direct administration into the affected area or its adjacent tissues may be necessary . the empirical establishment of the mechanisms which are exploited by the invention are described in detail in the following examples . these examples are illustrative of the utility of the present invention , but do not limit the invention . although the expression of tpa in the hippocampus and its induction by pharmacological and electrical stimulation are well - documented ( refs . 7 - 9 ), the role for the enzyme in the brain has not been established . since the four different cell types in the hippocampus - neurons , oligodendrocytes , astrocytes , and microglia - perform distinct functions which might involve proteolytic activity ( ref . 22 ), defining the site or sites of synthesis of tpa was an essential first - step in determining its function . the pattern of tpa mrna expression in the adult mouse hippocampus coincides with the pyramidal ( neuronal ) cell layer of the ca1 to ca3 regions of the hippocampal formation and the granule cell layer of the dentate gyrus ( ref . 7 ; and unpublished observations ). the intimate association of neurons with their surrounding glia , however , has previously made it difficult to determine unambiguously which cell type expresses tpa . transgenic mice ( tpa / lacz ) have been generated which carry a mouse tpa promoter fused to the bacterial lacz gene ( ref . 9 ). the expression pattern of β - gal in these mice generally reproduced endogenous tpa mrna expression in the hippocampus both in location and in transcriptional activation after induction of seizure . the resolution of cytoplasmic β - gal staining in the tpa / lacz transgenic mice made it possible to analyze brain sections for tpa promoter activity in great detail . a seizure - inducing agent , metrazol ( pentylene tetrazol ) ( 50 mg / kg ), was administered to tpa / lacz mice by intraperitoneal injection . after a recovery period of five days , animals were anesthetized and heart perfusion was performed using pbs followed by 4 % paraformaldehyde . the brains were removed and left in 30 % sucrose in fixative overnight at 4 ° c . coronal tissue sections ( 30 μm ) were prepared and stained overnight at 37 ° c . with x - gal ( ref . 23 ). when viewed under high magnification , the hippocampal β - gal staining showed a punctate pattern over the ca1 to ca3 regions and dentate gyrus ( dg ), but the size of the stained cells was smaller than the pyramidal cell bodies ( fig1 ). instead , the dimensions of the β - gal producing cells indicated that a subset of microglial cells surrounding the neurons was expressing β - gal and , by inference , tpa mrna . to more clearly exclude the potential contribution of neuronal activity , we eliminated neurons in adult tpa promoter / lacz mice by unilateral , intracerebral injection of kainic acid ( ka ). this excitotoxin is a cyclic , glutamate analog that can cause convulsions and neuronal degeneration . to assess neuronal cell death , brain sections were examined by cresyl violet staining of neuronal cell bodies . adult male mice , weighing approximately 25 g , were administered atropine ( 0 . 6 mg / kg of body weight ) by intraperitoneal injection , and then deeply anesthetized with metophane . the anesthetized mice were placed in a stereotaxic apparatus , and administered 1 . 5 nmol of kainic acid in 0 . 3 μl of pbs by unilateral injection into the hippocampus ( ref . 13 ). the coordinates of the injection were : bregma - 2 . 5 mm , medial - lateral 1 . 7 mm , and dorsoventral 1 . 6 mm . the kainic acid was delivered over 30 s , with the pipette maintained in place for an additional 2 min to prevent reflux of fluid . after a recovery period of 5 ( fig2 a ) or 16 days ( fig2 b or 2c ) brain sections were prepared as described in example 1 . the sections were mounted onto slides , dehydrated through increasing ethanol gradients , and then stained . in fig2 panel a was stained with cresyl violet , which stains neuronal cell bodies ; panels b and c were stained overnight for β - gal activity and counter - stained with neutral red . for the tpa enzymatic activity assay , wild - type mice administered kainic acid by injection were sacrificed 5 days after the injection . the brains were frozen and processed as described previously ( ref . 7 ), except that amiloride was not included in the overlay mixture . the photograph was taken under dark field illumination after 2 h incubation at 37 ° c . fig2 a - 2c illustrate the persistence of tpa / lacz - expressing cells following excitotoxin exposure . low magnification cresyl violet staining of coronal sections through the hippocampus illustrates the lesion generated by 1 . 5 nmol kainic acid ( ka ) on the ipsilateral side ( side of injection ; arrow ), whereas contralateral to the lesion no neuronal death was observed . fig2 a shows hippocampus from a wild - type tpa / lacz mouse 5 days after the injection . fig2 b shows tissue from a tpa / lacz mouse : higher magnification of the ipsilateral side , 16 days after injection . fig2 c shows tissue from a tpa / lacz mouse : higher magnification of the contralateral side , 16 days after injection . scale bar in fig2 b and 2c : 20 μm . note the persistence of the β - gal staining on the ipsilateral side where the pyramidal cells have been destroyed ( fig2 b ). given that neuronal cell death is observed within 12 hours following injection , and that the mice were examined at 16 days , it is unlikely that the β - gal staining represents residual , phagocytosed neuronal debris . fig2 d shows a tpa histoenzymatic assay on a coronal brain section of a wild - type mouse having had kainic acid injected unilaterally as in fig2 a . note the zone of proteolysis indicating tpa enzymatic activity primarily over the ca2 - ca3 hippocampal subfields and dg . the arrow points to the zone of proteolysis surrounding the injection track , where microglia accumulate . consistent with previous reports ( ref . 13 ), complete loss of neuronal cells in the ca1 to ca3 pyramidal cell layers was observed on the ipsilateral ( injected ) side ( fig2 a ). granule cells of the dentate gyrus were unaffected by the injection of ka . similarly , there was no noticeable degeneration on the contralateral ( uninjected ) side ( ref . 13 ). when adjacent tissue sections were stained for β - gal , the staining persisted on the ipsilateral side , even though no neuronal cells were present . in addition , the intensity of the staining in the absence of neurons was comparable to that of the unaffected , contralateral side ( fig2 b and 2c ). this result conclusively shows that tpa is not produced by neurons . after ka injection , a significant increase of β - gal expression was observed in the dentate gyrus on the ipsilateral side ( data not shown ). microglia have been reported to accumulate in the dentate gyrus after injury , even though neuronal death is not observed there ( ref . 14 ). since neuronal cell number increase is not observed in the dentate gyrus , this increase in β - gal staining is most likely due to accumulation of microglia . taken together , the results shown in examples 1 and 2 demonstrate that activated microglial cells are the major source of tpa in the hippocampus . having determined that tpa is a microglial protease , we investigated whether this enzyme plays a role in the degradation of neurons in the hippocampus . three different strains of mice were treated by injection of kainic acid : mice homozygous for a disrupted tpa allele ( tpa -/- ) ( ref . 24 ), and the two inbred , control strains ( c57 and 129 ) that were used to generate the tpa -/- animals . fig3 a includes two photomicrographs showing low magnification cresyl violet staining of coronal sections through the hippocampus . the injection of kainic acid was performed as described in example 2 . the brains of the treated mice were analyzed 5 days after injection . the left hand panel in fig3 a shows tissue from a wild - type 129 mouse ; the right - hand panel shows tissue from a tpa -/- mouse . the substantial destruction of the neurons in all of the ca fields in the control mouse ( fig3 a , left ) contrasts sharply against the extent of neuron survival in the tpa -/- mouse ( fig3 a , right ). the experiment was repeated with 8 c57 mice , 4 129 mice , and 12 tpa -/- mice . in control animals , essentially complete neuronal loss was observed in the ca1 to ca3 regions , similar to that of the tpa / lacz mice ( strain 129 , fig3 a , left ; strain c57 , data not shown ). by contrast , neuronal degeneration in the hippocampus of tpa -/- mice was minimal ( fig3 b , right ). ( these results are confirmed quantitatively in fig4 a .) although the degeneration observed in the tpa -/- mice was dramatically reduced , some cell loss was evident close to the injection site , suggesting that the lack of tpa increases the threshold necessary to effect degeneration . kainic acid is an agonist of one subtype of the glutamate receptor system , but there are other classes of glutamate gated ion channels : ( 2 - aminomethyl ) phenylacetic acid ( ampa ) and n - methyl - d - aspartate ( nmda ). to determine if the lack of neuronal loss observed was specific for the ka subtype of glutamate receptors , we injected intracerebrally agonists whose action is mediated through the ampa or nmda receptors . quisqualate ( 20 nmol ) and quinolinate ( 120 nmol ) were injected into tpa -/- mice as described in example 2 . the brains of the treated mice were examined 5 days after injection . fig3 b and 3c include photomicrographs comparable to those described for fig3 a , with tissue from the wild - type mouse in the left - hand panels and tissue from the tpa -/- mice in the right - hand panels . significant resistance to cell death was observed in tpa -/- mice after injection of quisqualate ( ampa receptors ) ( fig3 b ) or quinolinate ( nmda receptors ) ( fig3 b ). these experiments were repeated with 2 c57 and 2 tpa -/- mice for quisqualate , and 2 c57 and 2 tpa -/- mice for quinolinate . the data illustrated qualitatively in fig3 a - 3c were quantified as follows : two wild - type ( wt ) and two tpa -/- mice for each excitotoxin were treated and the tissue processed as described above . serial sections ( 30 μm ) were prepared and stained with cresyl violet . five or six matched sections from the dorsal hippocampus of wild - type and tpa -/- mice were drawn with a camera lucida and subjected to quantitative analysis ; the linear distances of spared ( intact ) ( black ), partly lost ( white ), and totally lost ( grey ) pyramidal cell layer were determined on each section . distances were digitized from the camera lucida drawings of the hippocampus . the measurements for each category over each hippocampal region were averaged across subjects in a group and plotted . fig4 a - 4c show the results of this analysis : kainate ( fig4 a ); quisqualate ( fig4 b ); and quinolinate ( fig4 c ). the linear distance of spared , partly degenerated , or completely lost neurons was measured in matched coronal sections at different rostrocaudal levels of the dorsal hippocampus . in each case the absence of tpa conferred dramatic resistance . for example , in ka - injected animals , c57 control mice demonstrated 66 % loss of ca1 , 38 % loss of ca2 , and 77 % loss of ca3 neurons . matched sections of tpa -/- mice showed no neuronal loss in ca1 or ca2 , and only 15 % loss in ca3 neurons . similar levels of resistance were observed with quisqualate and quinolinate . the results described in examples 3 and 4 implicate tpa as a key factor in the neuronal disappearance induced by excitotoxin . the decrease in neuronal cell death in tpa -/- mice is observed in relation to a wide range of excitotoxins . this implies that various glutamate receptor antagonists may be employed according to the method of the invention to regulate neuronal degeneration . the lack of neuronal degeneration in ka - injected tpa -/- mice could be due to a failure of microglial cell activation , which might result in neuronal persistence . to address this question , brain sections after ka injection were immunostained for the microglial - specific antigen f4 / 80 , which is produced only after activation ( ref . 25 ). kainic acid injection was performed as described in example 2 . the brain sections of the treated mice were immunostained with the activated microglia - specific polyclonal antibody f4 / 80 as described in example 1 . fig5 a - 5d are low magnification photomicrographs of f4 / 80 immunostaining of coronal sections through the hippocampus 5 days post - injection . fig5 a shows tissue of a 129 mouse ; fig5 b shows tissue of a tpa -/- mouse . maximal activation is observed on the ipsilateral side and around the injection site ( arrow ). microglia on the contralateral side are activated as well , but to a lower level . fig5 c is a high magnification micrograph of representative activated microglia in the ca1 field of stratum radiatum on the ipsilateral side of a 129 mouse . fig5 d is a high magnification micrograph of activated microglia of tpa -/- mouse . scale bar : 20 μm . qualitatively , microglia from all three mouse strains behaved similarly : the cells were activated around the injection site and along the pyramidal cell layers of ca1 to ca3 , whereas no activation was observed on the uninjected side ( fig5 a and 5b ) ( ref . 13 ). activated microglia were also observed in the deep portion of the dentate granule cell layer of the basket pyramidal cells . these cells are not susceptible to excitotoxicity and are thus spared from degeneration ( ref . 14 ). despite qualitative similarity , however , quantitatively , the extent of immunostaining observed in tpa -/- mice was decreased compared to that in c57 or 129 mice . the intensity of f4 / 80 staining suggests an approximately 2 - fold lower degree of microglial activation in tpa -/- mice . this difference in intensity is less dramatic than the difference in neuronal persistence in the hippocampal ca pyramidal fields . microglial activation can also be assessed by morphology . in the hippocampus of wild - type mice , resident microglia have a characteristic radially - branched shape ( ref . 25 ). after injection of ka , microglial cell number increases and their processes become increasingly arborized ( ref . 13 ). the activation of microglia in tpa -/- mice was evaluated using these morphological criteria . an attenuation in morphological changes was observed when tpa -/- mice were compared to c57 ( data not shown ) and 129 mice ( fig5 c and 5d ). all of these indicate that tpa is involved in the activation pathway of microglial cells . after intracerebral injection of ka , control mice underwent epileptic seizures in the immediate post - operative period , consistent with the reported effect of injection of the excitotoxin ( ref . 26 ). in contrast , at this dose of ka , the tpa -/- mice did not exhibit overt seizures . we investigated this observation further by determining the responses of these mice to increasing concentrations of seizure - inducing agents . metrazol , a convulsant drug that acts through a gaba receptor and increases the transcription of tpa in the hippocampus ( refs . 8 , 9 ), was injected intraperitoneally into tpa -/- and control mice at the indicated concentration . kainate was injected as described above into another group of tpa -/- and control mice . convulsive behavior as observed within five minutes from the time of injection in the c57 or 129 mice . the onset of seizure for tpa -/- mice usually occurred approximately 15 - 20 minutes after metrazol delivery . seizures were classified using the following five categories ( ref . 27 ): 1 , arrest of motion ; 2 , myoclonic jerks of the head and neck , with brief twitching movements ; 3 , unilateral clonic activity ; 4 , bilateral forelimb tonic and clonic activity ; 5 , generalized tonic / clonic activity with loss of postural tone . to control against potential bias in interpretation of mouse behavior , the labels indicating the mouse strains were removed from the cages and replaced by numbers . the behavior of the mice was monitored by non - biased , &# 34 ; blind &# 34 ; judges . the tpa -/- mice had a higher threshold with respect to both metrazol - seizure induction ( fig6 a ) and kainate - seizure induction ( fig6 b ) as compared with the control strains . this resistance to seizure induction was evident with respect to the dose of agent , as well as with respect to the time delay between drug delivery and the onset of seizure ( data not shown ). since one consequence of seizure is neuronal degeneration ( ref . 28 ), it is consistent that the tpa -/- mice are resistant to both processes . our data show that tpa -/- mice are resistant to neuronal degeneration and seizure after excitotoxin injection . the brains of the tpa -/- mice were examined in detail , and no obvious morphological abnormalities were detected ( ref . 24 , and unpublished observations ). therefore , it is probable that the observed effects are due to lack of expression of tpa in the adult hippocampus . however , embryonic development in the absence of tpa might cause subtle alterations in the cytoarchitecture or circuitry of the brain , which could result in the observed phenotypes . with this reservation in mind , it is interesting to consider the effect of tpa on neurodegeneration and seizure from several points of view : 1 , the role of microglia cells and their activation ; 2 , the molecular mechanism by which tpa influences degeneration ; 3 , the relationship of tpa to other mutations implicated in altered seizure susceptibility ; 4 , the possibility that tpa plays a role in normal hippocampal plasticity ; and 5 , possible insights into neurodegenerative diseases and their treatment . previous evidence indicates that microglial protease activity is in certain circumstances related to neuronal survival . specifically , transection of the optic nerve leads to degeneration of ganglion cells , and injection of protease inhibitors into the vitreous body retards this degeneration ( ref . 15 ). the results described above now strongly suggest that microglial protease activity is attributable to tpa , and that tpa is synthesized not simply in response to dying neurons , but is directly involved in the destruction of those neurons . the fact that microglial activation is reduced in tpa -/- mice could partially explain the lack of neuronal degeneration . this decrease could be due to a diminished response of the hippocampal cells to ka . in general , the activation mechanism of microglia cells is not well defined . it is known that microglial activation is blocked by the nmda receptor antagonist , mk - 801 ( ref . 14 ), and that the transcriptional induction of tpa is also inhibited by mk - 801 ( ref . 8 ). these results , along with the attenuated microglial activation in the tpa -/- mice , indicate that activation and expression of tpa may be related . the molecular mechanism by which tpa influences neuronal degeneration is not known . the only defined substrate for tpa is the zymogen plasminogen . plasminogen might be increased in the brain since injury results in a compromised blood - brain barrier ( ref . 29 ); alternatively , local synthesis of plasminogen is possible , since its mrna is detected in the brain ( ref . 7 ) and microglia in culture secrete plasminogen ( ref . 30 ). if plasminogen is present , a classical cascade could be generated that would greatly amplify the proteolytic potential and promote tissue remodeling . these considerations raise the issue of what the ultimate target for the proteolytic activity might be , and how this process might regulate neuronal survival . activated microglia in culture secrete neurotoxic molecules that may be responsible for the death of neurons after cns injury ( ref . 31 ). it is possible that tpa and / or plasmin mediate the synthesis or processing of molecules with neurotoxic properties . if so , tpa would have a dual role in affecting both microglial activation and the generation of neurotoxins , and its absence would result in dramatic persistence of neurons . our observation of the resistance of tpa -/- mice to seizure identifies this protease as a participant in the convulsive pathway that alters seizure susceptibility . there are other genetically - defined mutations that appear to reside in a single gene and which predispose mice to convulsions ( refs . 32 , 33 ). since seizure susceptibility is genetically complex ( refs . 34 , 35 ), it will be interesting to determine the extent to which the tpa gene interacts with other loci associated with inherited convulsive disorders . it has been hypothesized that the morphological changes that occur after kindling and seizures are an exaggerated form of the structural changes that take place during long - term potentiation and learning / memory ( ref . 2 ). in this context , although tpa -/- mice do not exhibit any severely abnormal phenotype ( ref . 24 ), evidence has been presented that they display deficits in spatial learning , as tested by the morris swimming navigation task ( ref . 36 ). therefore , it appears consistent that the deficiency for tpa could lead to both learning impairment and seizure resistance . urokinase - type plasminogen activator ( upa ), another form of plasminogen activator , is not normally found in the mouse hippocampus ( refs . 6 , 7 ). however , ectopic expression of this enzyme in the brain of transgenic mice results in compromised learning abilities ( ref . 37 ). this finding , along with the acquisition / learning deficits of the tpa -/- mice , suggests that a delicate proteolytic balance may be necessary to ensure both maintenance and appropriate modulation of neuronal connections , which are required for normal learning memory capacities . there is extensive neuronal degeneration in the hippocampus in various pathological situations , e . g ., in alzheimer &# 39 ; s disease , in ischemia of the brain due to reduced blood flow , and in epilepsy ( ref . 5 ). while apoptosis - related mechanisms can explain some of these pathologies , it has not been established yet if they are involved in ka - induced nerve cell death ( refs . 26 , 38 ). our work has now identified tpa as a necessary link in experimentally - induced neuronal degeneration and seizure , and reaffirms that over - expression of tpa activity could contribute to neuronal destruction in some of these diseases . in this respect , alzheimer amyloid β - peptide analogs have recently been found to stimulate tpa activity in vitro ( ref . 39 ), further suggesting that elevated protease activity may be related to this pathology . therefore , over - expression of tpa in the hippocampus might lead to an in vivo mouse model of neuronal degeneration and / or susceptibility to seizure . such a model would be useful for testing whether inhibitors of tpa might be used to prevent destruction and seizure , induced either by excitotoxins or inherited genetic mutations . such inhibitors constitute a new class of compounds with therapeutic and diagnostic activity . thus , while there have been described what are presently believed to be the preferred embodiments of the present invention , those skilled in the art will realize that other and further embodiments can be made without departing from the spirit of the invention , and it is intended to include all such further modifications and changes as come within the true scope of the claims set forth herein . 2 . baudry , m ., in adv . neurol . 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