The research of neurospecific proteins and lysosomal peptidehydrolases in frontal neocortex during forming conditioned reaction of active avoiding of rats

Dynamics of the activity of neuronal cell adhesion molecule (NCAM) and lysosomal cysteine cathepsins B, L, H was researched in frontal neocortex of rat brain during forming a conditioned reaction of active avoiding. The quantitative estimation of NCAM content in the neocortex membrane fraction was carried on by ELISA in 3, 7, 14 and 21 days after starting animals’ training. The dynamics correlation between the NCAM content increasing and cysteine cathepsins activity was obtained, especially for aminopeptidase cathepsin H during the process of memory engram forming in frontal neocortex of rat brain.

In tro duc tion.The prob lem of neurochemical and molec u lar mech a nisms of neu ro log i cal mem ory is one of the most ur gent tasks of med i cal and bi o log i cal in ves tiga tions [1].Neu ro log i cal mem ory is formed, kept, and re pro duced on dif fer ent lev els, start ing with mo lec u lar, supra mo lecu lar, subcelullar ones, and end ing with intercellular level, where syn ap ses, neuromediators, hor mones, and neurospecific pro teins mediate the interaction of neurons [2,3].
One of the types of cell con tacts is ad he sion in ter action, which takes place at the as sis tance of mem brane glycoproteins -mol e cules of cell ad he sion.Neuronal cell ad he sion mol e cule (NCAM) is a neurospecific pro -tein, pro vid ing cell ad he sion in in ter ac tions of neu ron-neu ron or neu ron-ma trix type [4].NCAM partic i pates in syn apse mech a nisms, which are the foun dation of learn ing and mem ory [5].A sig nif i cant con centra tion of this pro tein was de ter mined in pre-and post-syn ap tic mem branes of neu rons, which ev i dences to its par tic i pa tion in syn ap tic mod i fi ca tions, caused by neuronal and im pulse ac tiv ity.It is known that learn ing is based on the pro cesses, re lated to the in crease in the num ber of syn ap ses and par tic i pa tion of var i ous syn aptic and mem brane mech a nisms of plas tic ity in the as socia tive pro cess [3].Due to the ac tiv ity of ex traor di nary stim uli, along with de struc tive pro cesses in the or ganism there is ev i dent de vel op ment of proliferative ones, de pend ing on a sin gle com mon fac tor -the sta tus of lysosomal ap pa ra tus of the cell.In both types of processes the key po si tions are given to lysosomes.They play the role of ini tia tive ob ject and pro vide con trol over the prog ress of de struc tive dis or ders in the or ganism [1,4].The re la tion of ac ti va tion of lysosomal appa ra tus to patho log i cal changes in the or gan ism is com mon knowl edge.It is much harder to de ter mine the role of lysosomes in adap tive-re stor ing pro cesses, tak ing place due to ex traor di nary stim uli.While study ing neurochemical and mo lec u lar mech a nisms of neu ro log i cal mem ory, the com mon prac tice is to reg ister changes (mo lec u lar and/or cy to log i cal), ca pa ble of re main ing for a cer tain time, which oc cur in the process of learn ing, mem o riz ing, and ad ap ta tion to any type of in flu ence.This is an ur gent and com pli cated task, solv ing which would al low man ag ing adap tive re ac tions of the or gan ism, in clud ing memory [5].
An im por tant role in the for ma tion of long-term mem ory is given to the ex change of pro teins, nec es sary con stit u ents of which are pro cesses of pro te ol y sis and mod i fi ca tion [6].The lysosomal en zymes are known to par tic i pate in deg ra da tion of pro teins, which come together with axoplasmic flow, with sub se quent use of amino ac ids in the for ma tion of new pro teins in the synap tic site di rectly [7].About 65-80% of sol u ble lysosomal peptidohydrolases are cysteine peptidohydrolases [8], in clud ing the most ac tive onescathepsin B (EC 3.4.22.1),cathepsin L (EC 3.4.22.15), and cathepsin H (EC 3.4.22.19).Their bi o log i cal functions are re lated to in ter nal and ex ter nal cell pro te ol y sis and pro cess ing of pro teins and pep tides.The in crease in the level of pro te ol y sis is a sign of neuronal de gen er ation or dys func tion of brain ar eas of aged peo ple, e.g.ac ti va tion of cysteine cathepsin B was de ter mined at Alz hei mer's dis ease [9,10].The study on the fea tures of re stricted pro te ol y sis re ac tions al lows un der stand ing the role of cysteine cathepsins in the func tion ing of a com pli cated adap tive sys tem of the or gan ism, which includes the pro cesses of form ing, keep ing, and re produc ing mne monic traces taking into account the significance of specific brain formations, which play the decisive role in them [11].
To en hance the un der stand ing of neurochemical mech a nisms of neu ro log i cal mem ory, we re searched changes in the ac tiv ity of lysosomal cysteine cathepsins B, L, H and in the con tent of NCA in the fron tal neo cortex of rat brain dur ing the for ma tion of con di tioned reflex memory.
Ma te ri als and Meth ods.The re search was car ried out on 79 Wistar line rats, weight 180-230 g.To es timate the dy nam ics cor re la tion of cathepsins B, L, H activ ity and the con tent of neurospecific pro tein NCAM in the pro cess of form ing mem ory engrams we used the con di tioned ac tive avoid ance re sponse (CAAR) [3] as a model of mnestic re ac tions.The se lec tion of this model was caused by the pos si bil ity of test ing the sta tus of the pro cess of form ing mem ory engrams on each day of learn ing.Con di tioned ac tive-de fen sive skill was formed in Y-type lab y rinth with elec tri fied floor of com part ments.The light ir ri tant was se lected for con ditioned stim u lus and no ci cep tive electrostimulationfor un con di tioned re in force ment.The train ing of an imals was con ducted in six ses sions per week with 10 con nec tions of con di tioned sig nal to un con di tioned re in force ment un til reach ing the train ing cri te rion of 95% pass ing to the lit com part ment, which took place be fore the in tro duc tion of no ci cep tive stim u lus.The an i mals were de cap i tated in 2 hours af ter the train ing ses sions.All the op er a tions with brain tis sues were con ducted at 0-4°C.
The in di ces of form ing engrams of con di tioned reflex mem ory, NCAM con tent, and the ac tiv ity of lysosomal cysteine cathepsins B, L, H in fron tal neo cortex of con trol and lab o ra tory rats were ana lysed af ter 3, 7, 14, and 21 days of CAAR for ma tion.NCAM con tent was de ter mined in the mem brane frac tion of fron tal neocor tex us ing ELISA.We used the vari ant of in hib it ing by an ti gen [12], and showed NCAM con cen tra tion in µg/g of tis sue.The free ac tiv ity of cathepsins B, L, and H was de ter mined in 10% so lu tion of homogenates in 0.025 M tris-buffer, pH 7.4, con tain ing 0.15 m NaCl and 1 mM EDTA [6].The ac tiv ity of cathepsin B was stud ied by the break ing up of p-nitroanilide N,a-benzoyl-D,L-arginine (BAPA) (Fluka, Swit zerland) [13], the ac tiv ity of cathepsin L -by hy dro ly sis of 1% azocasein, denaturated by 3 M urea [14], and the activ ity of cathepsin H -by hy dro ly sis of 2-naphtylamide L-leucine (Leu-HA, Koch-Light Lab o ra to ries, UK) [15].
The spe cific ac tiv ity was de ter mined in 1 ml of incu ba tion mix ture with pre lim i nary in cu ba tion of en -zymes for 15 min in the pres ence of 2 mM of 2-mercapthoethanol and 2 mM Na 2 -EDTA.The ac tivity was shown us ing the fol low ing sub strates: BAPAin µmol of p-nitroaniline per 1 min per 1 mg of pro tein; Leu-HA -µmol of 2-naphtylamine per 1 min per 1 mg of pro tein; azocasein -in con ven tional units of ad sorption at 366 nm per 1 min per 1 mg of pro tein.Brad ford's method was used to per form the quan ti ta tive es ti ma tion of total protein in the samples [16].
Sta tis ti cal pro cess ing of re sults was per formed accord ing to [17].
Re sults and Dis cus sion.The re search proved the cor re la tion be tween the changes in con cen tra tion of the mem brane-bound form of NCAM and the free ac tiv ity of in ves ti gated cysteine cathepsins B, L, and H in frontal neo cor tex of rat brain dur ing the for ma tion of con ditioned reflex memory.
The specificities of dy nam ics of the con tent of neurospecific pro tein NCAM in the mem brane frac tion and of the free ac tiv ity of lysosomal cysteine cathepsins B, L, and H on the back ground of changes in neurospecific pro tein NCAM while form ing CAAR are pre sented in Fig ure (a, b, c, re spec tively).
The spec i fic ity of dy nam ics of the ac tiv ity of cathepsin B was its prob a ble de crease by 33 and 35% (p < 0.05) on the 3 rd and 7 th days of train ing (Fig ure , a) and its in crease by 57% on the 14 th day, com pared to the con trol.The ten dency to in creas ing the ac tiv ity of cathepsin B com pared to the con trol was ob served on the 21 st day of form ing CAAR.It is note wor thy that the in crease in free ac tiv ity of lysosomal cysteine cathepsins is an early sen si tive sign of changes in the sta bil ity of lysosomes mem branes [6].The anal y sis of the change in NCAM con cen tra tion tes ti fies to prob able in crease in the con tent of in ves ti gated neurospecific pro tein on the 3 rd day of train ing.It is the 3 rd day af ter the be gin ning of the ex per i ment, when there are much fewer mis takes of an i mals in pass ing to the dark compart ment of the lab y rinth, at the same time there are much more re ac tions of ac tive es cape from the no ci ceptive ir ri tant.Dur ing the fol low ing stage (7 th -21 st days) of form ing the con di tioned ac tive-de fen sive skill of rats, the NCAM con cen tra tion in the mem brane fractions of neo cor tex in creases by about 86% with max imal con tent on the 21 st day of train ing, end ing the forma tion of sta ble con di tioned re flex mem ory.It may ev idence to en force ment of mem ory engrams and pos si ble cod ing of in for ma tion into long-term mem ory.Learning and mem ory are im por tant fea tures of vari abil ity and plas tic ity of the ner vous sys tem [13].There is ev ident cor re la tion between the increase in expression and concentration of neurospecific proteins and the stages of memory formation.
The dy nam ics of cathepsin L ac tiv ity and NCAM con tent at the for ma tion of con di tioned re flex mem ory of rats is pre sented in Fig ure , b.It dem on strates the increase in the level of free ac tiv ity of cathepsin L by 43 and 77% af ter 3 and 7 days of CAAR for ma tion, re spectively, and the de crease by 30 and 28% on the 14 th and 21 st days of ob ser va tion, respectively.
The changes in the level of cathepsin H ac tiv ity and NCAM con cen tra tion in the fron tal neo cor tex while form ing engrams of con di tioned re flex mem ory are pre -112 DROZDOV À. L. ET.AL.
The dynamics of NCAM content and the level of free activity of cysteine cathepsins B (a), L (b), H (c) in the frontal neocortex of rat brain in the process of forming conditioned reflex memory: 1 -NCAM (µg/g of tissue), %; 2 (a) -cathepsin B (µmol of p-nitroaniline per 1 min per 1 mg of protein), %; 2 (b) -cathepsin L (conventional units per 1 min per 1 mg of protein), %; 2 (c)cathepsin H (mmol of 2-naphtylamine per 1 min per 1 mg of protein), %; M±m, n = 8; *p and **p < 0.05, compared to the control for NCAM and cathepsin, respectively sented in Fig ure , c.It was de ter mined that there is a re liable in crease in the level of free ac tiv ity of lysosome cysteine aminopeptidase (cathepsin H) at the in crease of ex pres sion of the neurospecific pro tein, start ing on the 3 rd day of CAAR for ma tion.Free ac tiv ity of cathepsin H in creases by 75% on the 7 th and 14 th days of train ing, com pared to the con trol.Our ob ser va tions are in good agree ment with the data on sig nif i cant re ac tivity of lysosomes of ner vous tis sue cells, cells of neo cortex of brain, in par tic u lar, re gard ing the for ma tion of mne monic traces.The high est level of ac tiv ity of cathepsin H in the fron tal neo cor tex of rat brain was reg is tered on the 21 st day of form ing con di tioned re flex mem ory with sim i lar changes in the dy nam ics of NCAM con cen tra tion.The data pre sented ev i dence to the fact that lysosomal cysteine pro teas es -cathepsins B, L, and H -are in ten sively re act ing to the pro cess of CAAR for ma tion, tak ing place in brain tis sues and partic i pat ing in adap tive re or ga ni za tion of pro tein me tab olism.This pro cess may be based on the de crease in the sta bil ity of lysosome mem branes, which results in the increase in the level of free activity of cathepsins in the brain cortex of laboratory rats.

Con clu sions.
There fore, the de ter mined specificities of the dy nam ics of the con tent of NCAM, par tic i pat ing in the reg u la tion of the pro cesses of synap tic re-mod el ling and, ac cord ing to Yamagata et al. [18], ca pa ble of launch ing syn ap tic plas tic ity and matur ing of syn apse struc ture, tes tify to the fact that the reli able in crease in NCAM con cen tra tion has im pact on the pro cesses of plas tic re-or ga ni za tion of syn ap tic connec tions in the fron tal neo cor tex of rat brain, start ing on the 3 rd day of CAAR for ma tion.The dy nam ics of the lev els of free ac tiv ity of lysosomal cysteine cathepsins on the back ground of changes in the NCAM con tent in the fron tal neo cor tex of rat brain, where ac tive-de fensive skill was formed, ev i dences to their phys i o log i cal and neurochemical role in the processes of learning and CAAR formation.