Aminopeptidasas y estrés oxidativo en las enfermedades neurodegenerativas
- Autores: Raquel Durán Ogalla
- Directores de la Tesis: Francisco Vives Montero (dir. tes.), Blas Morales Gordo (codir. tes.), Francisco Alba Araguez (codir. tes.)
- Lectura: En la Universidad de Granada ( España ) en 2008
- Idioma: español
- ISBN: 9788469183502
- Tribunal Calificador de la Tesis: Manuel Ramírez Sánchez (presid.), Blanca Gutiérrez (secret.), Francisco Javier Barrero Hernández (voc.), Marc De Gasparo (voc.), José Manuel Baeyens Cabrera (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: DIGIBUG
- Resumen
Aging presents an increasing challenge for developed societies. Diseases associated to aging are on the rise, and neurodegenerative diseases are among the most important. They produce a progressive disability, a remarkable social and economical problem of our society.
The most frequent neurodegenerative diseases are Alzheimer disease (AD) and Parkinsons disease (PD). This study was focused mainly on PD. PD is a progressive movement disorder characterized pathologically by the selective loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) and the presence of prominent cytoplasmic inclusions, called Lewy bodies (LB). PD prevalence increases from 1 percent at the age of 65 to 5 percent at 85, making age the main risk factor for this disease. Most of PD patients are thought to be sporadic; however, in a 5 to10% of cases, PD is believed to have a genetic origin, showing both recessive and dominant modes of inheritance (Onyango, 2008).
Although the mechanisms responsible for neurodegeneration in PD are largely unknown, mitochondrial dysfunction, oxidative stress and impairment in protein metabolism have been proposed as the three more important pathogenic factors. These tree ethiological factors are shared with AD and HD.
The first study relating mitochondria and PD was published in 1989, when a deficiency in electron transport chain protein complex I was identified in SNpc neurons taken from PD patients (Shapira et al., 2006). Afterwards, epidemiological studies proved the existence of inhibitors from mitochondrial complex I that cause experimental parkinsonism. The best known neurotoxic was MPTP (1-Methyl 4-phenyl 1,2,3,6 tetrahydropyridine). MPTP is a meperidine analog highly selective for DA neurons. In astrocytes, MPTP is transformed by the enzyme monoamine oxidase B (MAO-B) in its active metabolite, MPP+ (1-methyl-4-phenylpyridium ions), which is uptaken by DA transporter and inhibits mitochondrial chain complex I, producing neuronal death. In non human primates, MPTP also produced the formation of ¿-synuclein aggregates LB like.
The impairment of the electron transport chain in mitochondria originates an important decrease in the production of ATP and in all biological mechanisms depending on energy production. One main effect of this cellular energy decrease is the increase in reactive oxygen species (ROS) production, leading to oxidative damage (Keeney et al., 2006). The term ROS usually refers to the generation of superoxide anion (O2.-) which is produced during the reduction of water (H2O) in the electron transport chain. O2.- is enzymatically or spontaneously converted to hydrogen peroxide (H2O2) at rapid rates. In the mitochondria, H2O2 is removed by gluthatione peroxidase; however, significant amounts of H2O2 are still able to diffuse from the mitochondria to cytosol. The existence of high levels of H2O2 together with other ROS may also damage DNA, proteins and membrane lipids, leading to cellular death, mainly for apoptosis.
Due to the high rate of metabolism and the special DA metabolism that produces high amount of ROS, dopaminergic neurons are highly susceptible to oxidative stress,. DA is inactivated by the enzyme MAO in a reaction that yields significant amounts of H2O2. DA may also suffer autooxidation processes leading to the formation of quinones and semiquinones, which are toxic per se, and may lead to ROS generation. Furthermore, SNpc contains high iron concentrations, which binds DA and neuromelanin. Iron also reacts with lipids and by the Fenton reaction produces peroxidate lipids (LPO). For this reason, free radicals are considered the main cause of nigral degeneration (Koutsiliere et al., 2002).
The impairment in protein metabolism produces neuronal damage as well. A feature of PD is the generation of ¿-synuclein aggregates. ¿-Synuclein is part of a gene family that includes ß- and ¿-synucleins and synoretin. Although the function of ¿-synuclein is not well understood, this protein is located in the synaptic terminal, and it appears to have a role in vesicular traffic and neurotransmission. It has little or no detectable secondary structure, and hence ¿-synuclein is referred to natively unfolded. ¿-Synuclein is one of several proteins associated with neurodegenerative diseases . It presents a high tendency towards aggregation due to characteristics of the central hydrophobic region of ¿-synuclein, which tends to self-associate. This central region is different from that of synucleins other than ¿. The end product of ¿-synuclein aggregation is the formation of heavily insoluble polymers of proteins known as fibrils. It is thought that the fibrils of ¿-synuclein are the building block of LB; however, LB contains proteins other than ¿-synuclein, including neurofilaments and other cytoskeletal proteins (Cookson, 2005).
Other dysfunction in protein metabolism is the ubiquitin-proteasome system (UPS). The UPS is an intracellular system necessary for non lysosomal degradation of short life, misfolded, mutant, and oxidatively damaged proteins. Proteins to be degraded are first marked by covalent attachment of a polyubiquitin chain to a lysine residue of the substrate. The polyubiquitinated protein is then degraded by a large proteolytic complex, the 26S proteasome, in ATP-dependent reactions (Barzilai et al., 2003). Selective impairment of proteasomal activity and reduced expression of proteasomal subunits have been reported in nigral postmortem tissue from sporadic PD patients, Because of this, the existence of a direct impairment of UPS by ¿-synuclein was proposed. Pathogenic species of ¿-synuclein can directly bind to 20/26S proteasomal subunit, impairing proteolytic activity (Moore et al., 2005). These findings suggest that a dysfunctional UPS may underlay vulnerability and degeneration of nigral DA neurons in sporadic PD.
In addition to the above cited pathogenic factors, there are other secondary mechanisms involved in the pathogenesis of PD. One of them is the microglial activation. Inflammatory events that may initiate or aggravate neuronal damage have been reported in striatum, SNpc and other brain structures of PD patients (McGeer et al., 1988; Liu et al., 2003). Microglia contributes to the neurodegenerative process through the release of a variety of proinflammatory cytokines such as tumor necrosis factor ¿ (TNF-¿), interleukin-1ß (IL-1ß) and IL-6, interferon-¿ (INF-¿), as well as the upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). These factors exacerbate the degeneration of neurons (Whitton, 2007).
Likewise, Alzheimers (AD) and Huntingtons (HD) disease are two chronic, progressive and degenerative diseases. AD is considered the more prevalent of neurodegenerative diseases, with a frequency of 4.4 percent of affected people in the global population. The frequency increases with aging ans hence age is considered the main risk factor. Most of cases are thought to be sporadic. AD is clinically characterized by the progressive loss of memory and other cognitive skills, finally leading to acute dementia (Bird, 2008). Pathologically, there is a selective loss of medium and large pyramidal neurons, along with the formation of extracellular deposits termed senile plaques, composed mainly of ß-amyloid fibrils, and intracellular deposits termed neurofibrillary tangles, composed of microtubule-associated protein tau in its hyperphosphorylated form (Upadhya et al., 2007).
The last neurodegenerative disease analyzed in this study was HD. HD is a genetic disease, showing dominant mode of inheritance (Petrasch-Parwez et al., 2007). It shows a low prevalence, between 5-10 cases per 100,000. HD is characterized by involuntary movements (chorea), due to a prominent cell loss of striatal medium spiny neurons. It is also accompanied of personality changes, dementia and weight loss. The defective gene in HD contains a repeat expansion of trinucleotide CAG within its coding region that express a polyglutamine repeat in the protein huntingtin (htt). In general, the number of CAG repeats is correlated to severity of the disease and indirectly with aging. HD patients show a number of CAG repeats of about 40-50, while more of 70 are observed in early-onset or juvenile forms. The mutant htt tends to aggregate, originating insoluble polymers known as inclusion bodies (IB). These aggregates interfere with normal proteins by recruiting some of them into its matrix, affecting to cell processes (Valera et al., 2007).
The pathogenesis of AD and HD shows similarities to those described in PD: mitochondrial dysfunction and oxidative stress (Zhu et al., 2004; Trushina et al., 2007; Klepac et al., 2007), protein aggregation (Higuchi et al., 2005; Spires et al., 2007; Valera et al., 2007), inflammatory phenomena (Li et al., 2004; Rojo et al., 2008) and apoptosis (Gil et al., 2008). However, it may be suggested that these disorders, PD, AD and HD, although with different etiology and clinic, share at least part of pathological mechanisms that lead to selective neuronal loss and neurodegeneration.
These diseases are progressive, and an early diagnosis is a desideratum. Thus, many researchers are looking for biomarkers that could predict the disease before the symptoms appear. In PD, this effort has been directed to search for oxidative stress, because it is one of the main mechanisms implicated in its pathogenesis (Serra et al., 2001; Sohmiya et al., 2004; Prigione et al., 2006), together with the analysis of alpha-synuclein concentration (El-Agnaf et al., 2006; Lee et al., 2006).
A common feature of neurodegenerative diseases the impairment of protein metabolism. In the brain, there is an abundant group of enzymes related to protein and peptide metabolism known as aminopeptidases (APs). Previous research showed that patients suffering from PD and animal models with induced experimental parkinsonism presented significant changes in AP activities (Banegas et al., 2005, 2006). In the striatum, neuropeptides such as cholecystokinins, angiotensins, and enkephalins coexist together with DA. The role that APs play in the striatum and in the physiopathology of the neurodegenerative diseases that affect to the nigrostriatal system is largely unknown. Besides, there are a large number of striatal fibers that contain cholecystokinin, which come from prefrontal cortical areas and modulate autonomic and endocrine functions. Once they are released to synaptic terminal and activate their respective receptors, these neuropeptides are hydrolyzed mainly by membrane-binding peptidases. Here we focus on the group of the APs, which are the most abundant proteolytic enzymes in the brain, and play a key role in controlling small bioactive peptides functions. Hence, it is highly relevant to study the enzymatic activities of APs in PD and to analyze their contribution to the onset and progression of the disease.
Banegas et al. (2005) observed a marked decrease in these aminopeptidase activities in some brain areas (striatum, substantia nigra, and prefrontal cortex) in unilaterally-lesion rats with neurotoxic 6-hydroxydopamine (6-OHDA). Later, and on the basis of these results, they analyzed alanine-, cystine-, aspartate-, and glutamate-aminopeptidase activities (aaAPs) in plasma from PD patients. They found a significant decrease in all AP activities except for Ala-AP (Banegas et al., 2006). Similar results were found in human brain (Mantle et al., 1995), even though these could not be compared with those obtained from plasma of lesion rats, in which the activities changed depending on the hemisphere where the lesion was performed (Banegas et al., 2004). Nevertheless, these results found in experimental animal models produce an acute pathology different to PD in human patients.
In addition to these functions, the decrease in the AP activities has been related with a decrease in the catalytic processes described in the neurodegenerative diseases, which lead to the formation of protein aggregated (LB, inclusion bodies, senile plates, etc,.). In AD, the APs have a direct implication in the catabolism of peptide A-beta. Recently, it has been demonstrated that these deposits are not the consequence of an overproduction but rather to a reduction in the degradation processes (Saido, 1998). The possibility that these enzymes may participate in the peptide A-beta catabolism was supported by the structural analysis of the amyloid deposits, finding a higher heterogeneity in the amino-terminal ends as a consequence of the AP activity (Kuda et al., 1997; Saido, 1998). The candidate enxime seems to be glutamate aminopeptidase (GluAP), showing the lowest activity in plasma from AD patients, stimulating the accumulation on insoluble and pathogenic structures (Kuda et al., 1997). However, the action of the APs does not exclude the participation of other peptidases and proteases in the catabolism of Aß.
This evidence has led us to evaluate the role of the APs in the neurodegenerative diseases, mainly in PD. However, it is also interesting to analyze if a similar pattern exists in AD and HD, which could be expected due to the parallelism observed in the molecular mechanisms responsible of the pathogenesis. Given that in the three previously mentioned diseases both the neuronal death and the insoluble aggregates formation are progressive, it is possible that the APs activities were modified during the progression of the disease. Therefore, this analysis may also provide useful information about the stage of the disease in the patient.
We have also evaluated the importance of the oxidative stress phenomena on the neurodegeneration. Mitochondrial dysfunction and electron transport chain impairment are the main responsible mechanisms of ROS production (Trushina et al., 2007; Sayre et al., 2008). An analysis of oxidative stress evolution may provide a better understanding of its implication in neurodegenerative processes. Furthermore, there is great controversy about antiparkinsonian treatment both in the progression and aggravation of PD, specially referring to the role of levodopa. We previously reported the antioxidant and protective roles of levodopa against lipid peroxides formation in blood plasma of PD patients (Agil et al., 2006), which was subsequently confirmed by other researchers (Prigione et al., 2006). However, the redox status of PD patients prior to the treatment was not analyzed in any case. Besides, this study may allow us to test, on the one hand, whether oxidative stress is aggravated with PD progression and, on the other, if levodopa modifies the basal oxidative status after specific PD treatment.
Thus, this study has two main goals: i) to study the possible implication of APs in the pathology of PD, AD, HD; ii) to analyze the relationship between oxidative stress and neurodegenerative diseases; and, iii) to investigate if specific treatment changes APs activities and oxidative stress in the case of PD patients.
MATERIAL AND METHODS Subjects Subjects in this study were divided in six different groups. First, we formed a control group that consisted of a randomized group of sixty healthy subjects (C group). Second, there were forty two patients who were diagnosed of sporadic Parkinsons disease, all of them under treatment for at least one year before collection of blood samples (PDt group). Third, we grouped fifty three patients who were also diagnosed of sporadic PD but started specific treatment after the collection of blood samples for this study (PDnt group). In a fourth group there were twenty eight patients diagnosed of Alzheimers disease (AD group). Fifth, we formed a group with thirty four patients were diagnosed of Huntingtons disease (HD group). A final group was composed of forty patients with ischemic ictus disease (Ic group). All patients were diagnosed by at least two neurologists in the Neurology Unit of the University Hospital of Granada, following the suitable criteria in every case. This study was approved by the Ethical Committee for Human Research from the same hospital.
Material Aminopeptidase enzymatic assays Alanyl-, aspartyl- and leucinyl-2-naphthylamide substrates, 2-naphthylamide, bovine serum albumin, dithiothreitol (DTT), manganese chloride, brilliant blue G and trizma base were obtained from SIGMA. Cystinyl-di-2-naphthylamide substrate and di-sodium hydrogen phosphate dihydrate were obtained from FLUKA. Glutamyl-2-naphthylamide substrate was obtained from BACHEM. Dimethyl sulfoxide (DMSO), hydrochloric acid, acetic acid, sodium acetate, ortho-phosphoric acid, ethanol absolute were obtained from PANREAC. Finally, methionin, calcium chloride, sodium dihydrogen phosphate were obtained from MERCK.
Non-denaturing electrophoresis and SDS-PAGE/Immunoblotting Proteo Prep immunoaffinity columns, leucine aminopeptidase (CD13), sodium dodecyl sulfate, Fast black K salt (FBK), tween 20, bovine serum albumin and bradford reagent were obtained from SIGMA. Rabbit policlonal IgG CD13 antibody, goat aminopeptidase A antibody, donkey anti-goat IgG-HRP antibody were obtained from Santa Cruz Technologies. Aminopeptidase A/ENPEP recombinant human protein was obtained from R&D System. Anti-rabbit IgG-HRP antibody and ECL-plus western blotting detection system were obtained from Amersham. Prestained SDS-PAGE standard broad range, bis-acrylamide solution (30% Acrylamide/Bis, solution 29:1), immuno-blot PVDF membrane, mini-protean III system and trans-blot semi dry transfer cell were obtained from Bio-Rad. Tetramethyl ethylendiamin (TEMED) was obtained from MERCK. Medical X-ray film and developing system CURIX 60 were obtained from AGFA.
Determination of plasma lipid profile Cholesterol CHOD-PAP, Triglycerides GPO-PAP and HDL-cholesterol plus 2end generation kits were obtained from ROCHE.
Determination of plasma lipid peroxides and lactic acid levels PeroxiDetect and L-lactic acid Enzymatic Bioanalysis kits were obtained from SIGMA and ROCHE, respectively.
Determination of plasma ¿-synuclein level Human ¿-synuclein Immunoassay kit was obtained from Invitrogen.
Methods Collection of blood plasma Blood samples were extracted in the cubital vein, in tubes containing EDTA as anticoagulant. Plasma was isolated by centrifugation for 15 min at 3,000 r.p.m. and stored at -80 ºC until analysis.
Aminopeptidase enzymatic assay Alanyl- (AlaAP), cystinyl- (CysAP), aspartyl- (AspAPA), glutamyl- (GluAP) and leucinyl- (LeuAP) aminopeptidase activities in plasma were measured fluorometrically in duplicate using L-Ala-ß-naphtylamide (L-AlaNNap), L-Cys-di-ß-naphtylamide (L-CysNNap), L-Asp-ß-naphtylamide (L-AspNNap), L-Glu-ß-naphtylamide (L-GluNNap) and L-Leu-ß-naphtylamide (L-LeuNNap), respectively as substrates in agreement with Greenberg (1962), modified by Ramírez et al. (1997) for AlaAP, Tobe et al. (1980) for GluaAP and Cheung and Cushman (1971) for AspAP. All the reactions were stopped by adding 0.1 ml of 0.1 mol/l of acetate buffer, pH 4.2. The amount of ß-naphthylamine released as a result of the aminopeptidase activity was measured fluorometrically at 410 nm emission wavelength with an excitation wavelength of 340 nm. The sensitivity of the method allows to measurements of AP in the pmol range. Proteins were quantified in duplicate by the method of Bradford, using BSA as standard. Specific aminopeptidase activities were expressed as pmol of AlaNNap, CysNNap, AspNNap, GluNNap and LeuNNap hydrolyzed per min per mg of protein. Fluorogenic assays were linear with respect to time of hydrolysis and protein content.
SDS-PAGE and Immunoblotting of alanine and glutamate aminopeptidases Previous to SDS-PAGE and Immunoblotting, plasma samples were subjected to depletion of the albumin and IgG proteins by immunoaffinity columns, according to the protocol supplied in the kit.
Afterwards, 20 ¿l of plasma containing 90 and 75 ¿g of total proteins by Bradford method were subjected to 12% SDS-PAGE. After electrophoresis, proteins were transferred from gel to a PVDF membrane in a buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol, using a semidry system of transference, at 100 mA, 20 V for 30 min. Blotting of the membrane was performed after blocking non specific binding with 5% free fat milk and 0.1% Tween 20 in Phosphate-buffered saline (PBS) pH 7.5 overnight. After three washings with PBS-Tween at 0.1%, the blot was incubated with the specific primary antibody at dilution 1:1000 in PBS containing 0.5% milk, at 4°C for 60 min. After several washings with PBS-Tween at 0.1%, the blot was incubated for 1 hour with anti-goat or anti-rabbit IgG horseradish peroxidase conjugated antibodies, diluted 1:5000 or 1:2000, respectively. Finally, after three washings, the blot was visualized using enhanced chemiluminescence detection system according to the suppliers instructions and CURIX 60 developing system.
Non denaturing PAGE from alanine and leucine aminopeptidases In order to detect the enzymatic activities from isolated aminopeptidases, 20 ¿l of plasma containing 400 and 200 ¿g of total proteins by Bradford method, were subjected to 10% non-denaturing PAGE. After electrophoresis, gel was incubated with the specific substrate of the enzyme (aminoacyl-ß-naphthylamide) in phosphate buffer 0.1 M pH 6.1 containing 0.15 % Fast Black K salt (FBK salt) at 37º C for 30 min. The enzyme, after breaking down the specific substrate, liberates ß-naphylamide which reacts with the salt originating a purple dye. After, gel was washed for 30-40 minutes using 25 % acetic acid, so that dyed strips correspond with the sites where enzyme is located and the colour intensity is proportional to enzymatic activity.
Determination of plasma lipid profile Total cholesterol, triglycerides, HDL cholesterol levels were measured according to the protocols supplied in each kit, in a Roche/Hitachi automatic analyzer of clinical chemistry from the unit of Clinical Analysis of the University Hospital of Granada. LDL cholesterol level was calculated by the formula of Friedewald. Alls levels were expressed as mg/dl.
Determination of plasma lipid peroxides and lactate Plasma lipid peroxides and lactate were measured in duplicate according to the protocols supplied in each kit. The levels were expressed as nmol/ml and g/l, respectively.
Determination of plasma ¿-synuclein Plasma ¿-synuclein level was measured in duplicate according to the protocol supplied in the kit. The level was expressed as ng/ml.
Statistical methods To reach different targets of the theses a statistical analysis was carried out in the following steps:
I. Descriptive statistics for all variables of the study using summary measures (mean, standard deviation, percentiles, etc) when variables are quantitative and frequency distribution when categorical variables.
II. One way analysis of variance was used to study differences among groups by age and pairwise comparisons with Bonferroni¿s penalization if that was significant. Fisher exact test extension to rxc tables was used to study association between group and gender of the patient. Finally association between pairs of quantitative variables was studied using a scatter plot to discard nonlinear association and Pearsons correlation coefficient.
III. One way analysis of variance was carried out to detect differences among groups for each variable of the study, and when significant pairwise comparisons with Bonferronis penalization using Newman-Keuls method was used. Nevertheless this was not an appropriate method because groups were different by age and gender of patients; so analysis of covariance was used to study differences among groups adjusted by age and sex; when analysis of covariance was significant pairwise comparisons were carried as previously said. To reach homogeneity of variances neperian logarithm transformations was applied.
STATA 10.1 was used for computations. A signification of 0.05 was selected for all significance tests (without or with penalization).
RESULTS Table 1 shows the distribution of the subjects by age and sex. There were significant differences in age between groups (P < 0.001), probably due to the different onset of each disease. Also, it was not possible to select aged people that not were under treatment (antidiabetics, antihypertensives, etc.). After statistical analysis controlling the possible differences due to age and sex among the subjects for this study, no difference was found between groups. Table 2 shows medication regime of PDt group. In this group, all patients were under treatment with L-dopa plus carbidopa. Plasma lipids of all subjects were analyzed, and Table 3 shows total cholesterol, triglycerides, HDL cholesterol and LDL cholesterol. All groups were normolipemic, except Ic group that showed total cholesterol, HDL and LDL cholesterol significantly lower than others groups. This difference may be due to the fact that a 30 percent of Ic group was under treatment with statines.
GROUP n AGE SEX C 60 48.02 ± 2.38 Male 30 Female 30 PDt 42 65.36 ± 1.39 Male 28 Female 14 PDnt 53 64.26 ± 1.59 Male 28 Female 25 EA 28 75.71 ± 0.95 Male 11 Female 17 EH 34 46.35 ± 2.22 Male 14 Female 20 Ic 40 76.10 ± 1.75 Male 27 Female 13 TOTAL 257 61.33 ± 1.08 Male 138 Female 119 Table 1. Distribution by age and sex of groups used in this study. Mean ± SEM.
DRUG n % CASES Levodopa 26 72,22 Carbidopa 26 72,22 Pramiprexol 17 47,22 Entacapone 12 33,33 Cabergoline 6 16,67 Rasagiline 5 13,89 Selegiline 5 13,89 Table 2. Medication regime of PDt group: distribution of drugs with major consumption. Values represent frequency and percentage of cases.
GROUP n TOTAL CHOL. TRIGLYCERIDES HDL CHOL. LDL CHOL.
C 51 191.039 ± 4.5188 109.902 ± 8.3763 55.872 ± 2.2768 114.747 ± 4.1011 PDt 42 177.810 ± 4.7150 129.310 ± 10.0126 50.554 ± 2.0842 106.081 ± 4.2378 PDnt 54 183.833 ± 4.8824 122.815 ± 7.4097 51.464 ± 2.2922 112.769 ± 4.3166 AD 28 202.357 ± 8.4748 140.821 ± 19.3921 53.296± 3.9497 111.361 ± 4.6483 HD 34 182.394 ± 8.5628 122.333 ± 12.1816 50.939 ± 2.9297 114.070 ± 4.1895 Ic 40 153.650 ± 5.9010 134.525 ± 11.9358 35.620 ± 2.9171 87.532 ± 4.7074 Table 3. Plasma levels of total cholesterol, triglycerides, HDL cholesterol and LDL cholesterol (mg/100mL). Mean ± SEM.
Plasma aminopeptidase activities Figure 1 shows aminopeptidase plasma activity for each group. There were no differences between control and patient groups in AlaAP plasma activity. However, AlaAP activity was higher in PDnt group, compared to PDt and Ic group (P < 0.05) (Fig. 1A). CysAP plasma activity was lower in PDt and Ic groups, compared to C group (P < 0.05). The comparison between PD groups showed a significant higher CysAP activity in PDnt respect to PDt group (P < 0.01). Also, CysAP activity was lower in Ic group than in PDnt (P < 0.001) and AD groups (P < 0.05) (Fig 1B). AspAP plasma activity was lower in HD and Ic groups compared to C group (P < 0.05 and P < 0.01, respectively). AspAP plasma activity was higher in PDnt and AD groups than in PDt group (P < 0.001). AspAP activity was also higher in PDnt group than HD and Ic groups (P < 0.001 for both groups) and higher in AD group respect to HD (P < 0.001) and Ic (P < 0.05) groups (Fig. 1C). GluAP plasma activity was significantly lower in HD group compared to C group (P < 0.01), PDnt group (P < 0.001), AD group (P < 0.001) and Ic group (P < 0.05) (Fig. 1D). Finally, LeuAP plasma activity was higher in PDnt and AD groups respect to PDt (P < 0.01 for both groups). On the contrary, LeuAP activity was lower in HD and Ic groups respect to PDnt group (P < 0.01 and P < 0.001, respectively) and also respect to AD group (P < 0.05 and P < 0.001 respectively) (Fig. 1E).
Plasma aminopeptidase activities related to medication regime in Parkinsons disease AlaAP, CysAP, AspAP and LeuAP plasma activities were significantly lower in PDt group than in PDnt group. To test if these differences were due to antiparkinsonian treatment and test the possible influence of drugs showed in Table 2, aminopeptidase activities were analyzed statistically. The enzymatic activity of AlaAP and CysAP was similar in patients with and without medication. AspAP activity was not dependent on medication, except in those under treatment with cabergoline (P < 0.01), rasagiline (P < 0.01) and selegiline (P < 0.05), among which activity was lower. GluAP activity also remained stable regardless of treatment, except for rasagiline (P < 0.001), which was related to lower activity. Finally, LeuAP activity did not change significantly in relation to medication either, except in the case of those treated with cabergoline (P < 0.01), rasagiline (P < 0.01) and selegiline (P < 0.05) for whom activity was lower.
Isolation and detection of plasma aminopeptidases by SDS-PAGE/Immunoblotting and non denaturing PAGE The results showed in this part correspond to qualitative analysis of aminopeptidases best recognized. Blood plasma contains very low levels of these proteins and thus it is very difficult to detect them. To avoid cross reactions between aminopeptidases and plasma proteins, the most abundant proteins, albumin and immunoglobulins, were extracted by immunaffinity columns. Plasma samples of next assays were taken from different subjects.
An example of Western blot of AlaAP from plasma of subjects belonging to groups C and PDnt is shown in Fig. 2. From left to right, the first two strips correspond to 75 and 50 nanograms, respectively, of commercial AlaAP with 140 kDa of molecular weight. The next two strips are AlaAP contained in 90 and 75 micrograms of plasma proteins, respectively, of a subject of C group. The last two strips are AlaAP in similar samples of a PDnt subject. AlaAP concentration in the PDnt patient was higher than in the C subject.
Figure 2. Western blot of AlaAP from blood plasma of C and PDnt subjects.
GluAP Western blot from blood plasma of C and PDnt subjects is shown in Figure 3. 170 nanograms of commercial GluAP were used to electrophoresis together with blood plasma samples, but it could not be detected in the respective strip. From left to right, the first two lines correspond to GluAP contained in 90 and 75 micrograms, respectively, of plasma proteins of a C subject. The last two strips are the GluAP contained in similar amounts of a PDnt subject. Molecular weight of GluAP is approximately 130 kDa. GluAP concentration was higher in PDnt patient than in C subject.
Figure 3. Western blot of GluAP from plasma of C and PDnt subjects.
Figure 4 shows a Western blot of AlaAP from plasma of C and HD subjects. From left to right, the first two strips correspond to 150 and 100 nanograms, respectively, of commercial AlaAP, with a molecular weight of 140 kDa. The next two strips are AlaAP contained in 90 and 75 micrograms, respectively, from plasma proteins of a C subject. The last two strips correspond to AlaAP contained in similar amounts of a HD subject. AlaAP concentration was lower in HD subject than in C subject.
Figure 4. Western blot of AlaAP from plasma of C and HD subjects.
Western blot of plasma GluAP from C and HD subjects is shown in Figure 5. As in Figure 3, the strip with commercial GluAP could not be revealed. From left to right, the first two lines are GluAP restrained in 90 and 75 micrograms, respectively, of plasma proteins of a C subject. The last two strips correspond to GluAP restrained in similar sample amounts of a HD subject. The approximate molecular weight of GluAP is 130 kDa. GluAP concentration was lower in HD subject than in C subject.
Figure 5. Western blot of GluAP from plasma of C and HD group subjects.
Figure 6 shows a blot of AlaAP from plasma of C and PDnt subjects, using non denaturing gel electrophoresis. Band intensity is proportional to enzymatic activity. From left to right, the fist four strips correspond to AlaAP activity displayed by 400 and 200 micrograms (both in duplicate) of plasma proteins of a C subject. The next four bands show AlaAP activity of similar sample amounts (in duplicate) taken from a PDnt subject. AlaAP activity was higher in PDnt subject than in C subject.
Figure 6. Non denaturing gel electrophoresis from plasma of C and PDnt subjects incubated with specific substrate of AlaAP.
Figure 7 shows a blot of LeuAP from plasma of C and PDnt subjects, using non denaturing gel electrophoresis. Band intensity is proportional to enzymatic activity. The first four bands are, left to right, LeuAP activity displayed by 400 and 200 micrograms (both amounts in duplicate) from plasma proteins of a C subject. The following four bands correspond to LeuAP activity displayed in similar amounts of a PDnt subject. LeuAP activity was higher in PDnt subject than in C subject.
Figure 7. Non denaturing gel electrophoresis from plasma of C and PDnt subjects incubated with specific LeuAP substrate.
Figure 8 shows a blot of AlaAP from plasma of C and HD subjects in non denaturing gel electrophoresis. Band intensity is proportional to enzymatic activity. From left to right, first four lines are AlaAP activity displayed in 400 and 200 micrograms sample (both amounts in duplicate) from plasma proteins of a C subject. The next four lines are AlaAP activity displayed in similar sample amounts of a HD subject. AlaAP activity was higher in C subject than in HD subject.
Figure 8. Non denaturing gel electrophoresis of plasma of C and HD subjects incubated with specific AlaAP substrate.
Figure 9 shows a blot of LeuAP from plasma of C and HD subjects. Band intensity is proportional to enzymatic activity. From left to right, the fist three lines are LeuAP activity displayed in 400 (in duplicate) and 200 micrograms, respectively, from plasma proteins of a C subject. The next three lines are LeuAP activity displayed in similar amounts of a HD subject. LeuAP activity was higher in C subject than in HD subject.
Figure 9. Non denaturing gel electrophoresis from plasma of C and HD subjects incubated with specific LeuAP substrate.
Lipid peroxides and lactate concentration in plasma Plasma lipid peroxides (LPO) and lactate (LA) concentrations in control and patients groups are shown in Figure 10. We observed that A. Lipid peroxides were significantly lower in C group than in PDt (P < 0.001), PDnt (P < 0.001), HD (P < 0.01) and Ic (P < 0.01) groups. The lowest concentration was found in AD group, although there was not significant difference compared to C group. In patient groups, lipid peroxides were lower in AD than in PDt, PDnt, HD and Ic groups (P < 0.001). B. Plasma lactate was also lower in C group than in PDt (P < 0.01) and Ic (P < 0.001) groups. There were not significant differences among patient groups.
Plasma ¿-synuclein concentration in PD groups and its correlation with plasma aminopeptidase activities Figure 11 shows plasma ¿-synuclein concentration in groups C, PDt and PDnt. Plasma ¿-synuclein was higher in PDt and PDnt groups than in C group (P < 0.05).
Figure 10. Plasma lipid peroxides and lactate concentrations of control and patients groups. Plasma lipid peroxides in nmol/mL and lactate in g/L. a: differences respect to C group; b: differences respect to PDt group; c: differences respect to PDnt group; d: differences respect to AD group. Mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.
Figure 11. Plasma ¿-synuclein in groups C, PDt and PDnt. Concentration in ng/mL. a: difference respect to C group. Mean ± SEM. * P < 0.05.
It is possible that ¿-synuclein was hydrolyzed by aminopeptidases. If this was the case, it would be interesting to study the correlation between aminopeptidases and plasma ¿-synuclein. However, in this study we did not found any correlation between them (Table 4).
GROUP AlaAP CysAP AspAP GluAP LeuAP C r -0,0198 0,0826 -0,1578 0,0210 -0,1733 P 0,8962 0,5852 0,2949 0,8900 0,2493 PKct r -0,1765 0,0329 0,1378 0,1918 0,1611 P 0,2761 0,8403 0,3963 0,2358 0,3206 PKst r 0,0352 -0,0717 -0,0704 -0,0544 -0,0799 P 0,8041 0,6135 0,6200 0,7019 0,5733 Table 4. Study of the possible correlation between plasma ¿-synuclein concentration and plasma AP activity in C, PDt and PDnt groups. r: coefficient of correlation.
P: significance.
DISCUSSION One main feature of neurodegenerative diseases (PD, AD, HD) is a severe alteration in protein metabolism. Aminopeptidases constitute an important group of enzymes related to peptide and protein metabolism; for this reason, it is relevant to study their changes in activity in neurodegenerative diseases. Of these diseases, the only that has specific treatment is PD. Thus, we divided PD patients in two groups, without and with treatment, to assess if medication changes aminopeptidases activity. Our results showed that PDnt group presented a higher AP activity in plasma than PDt and this group showed, in general, similar activity than controls (lower activity in the case of CysAP). This suggests that PD cause an increase plasma AP activity and PD medication normalize the activity of these enzymes. Banegas et al. (2006) found similar results CysAP activity in treated PD patients although this is the first time that AP activity is studied in early diagnosed PD patients without treatment. The influence of medication among subjects in the PDt group on AP activity was also individually analyzed. After comparing subjects in the PDnt group for major consumption drugs as levodopa, carbidopa, pramipexol and entocapone, we did not find significant differences. However, we found a decrease in AspAP and LeuAP activities in PDt patients taking cabergoline, rasagiline and selegiline, and a lower GluAP activity in PDt under treatment with rasagiline compared to non-taking patients. We conclude that, altogether, antiparkinsonian drugs contribute to normalize APs activity, as it has been explained above.
Dopaminergic synaptic terminals of basal ganglia release DA together with several peptides, all these being potential substrates of APs such as cholecystokinins, angiotensins and enkephalins (Waters et al., 1995). Although the role of these peptides in neurotransmission is poorly understood, it appears that they act as neuromodulators and are necessary for the correct functioning of the basal ganglia circuits. It has been reported that other monoaminergic transmitter system, serotonine, APs is important in the interaction with several peptides in serotoninergic synapsis. Low AP activity has been observed in animals that up-express serotonin in rat brain (Cicin-Sain et al., 2008).
Besides characteristic motor symptoms, PD patients also have a high prevalence of autonomic dysfunctions, specially a decreased sympathetic innervation (Li et al., 2002), indicating that neurodegeneration also affect autonomic system neurons. In synapses of autonomic ganglia, many substrates of APs coexist with acetylcholine (ACh). Changes in AP activity may alter metabolism of the peptides released together with Ach. Also, changes in plasma AP activities may be related with autonomic alterations that occur in PD. In addition to autonomic dysfunction, changes in the hypothalamus-pituitary axis have been reported in PD. Thus, many studies have shown disturbances in the hormonal secretion from PD patients, such as reduction of somatostatin and ß-endorphin in cerebrospinal fluid, Met-enkephalin in mesencephalon, putamen and pallidum, and Leu-enkephalin in striatum (Sandyk et al., 1987). Moreover, a substantial loss of orexin and melanin-concentrating hormone cells was detected in dorsomedial and perifornical hypothalamic regions from PD patients (Thannickal en al., 2007). Moreover, it was reported that DA therapy may affect the pituitary function through a decrease in prolactin secretion and an increase in growth hormone secretion (Schaefer et al., 2008). Several APs were identified in the CNS that inactive endogenous opioid peptides (Hui, 2007). APs was also reported to play a main role in the regulation of circulating hormones such as angiotensins and others blood pressure-controlling hormones and renal function (Banegas et al., 2006; Speth et al., 2008). Recently, it was proposed other functions for angiotensins that may be regulated by APs. Ang IV promotes DA liberation in striatum (Stragier et al., 2007) and improves cholinergic transmission in rat hippocampus and other regions related to memory and cognitive functions (Albiston et al., 2003; Albiston et al., 2004). Ang II may stimulate the generation of ROS activating the mitochondrial enzyme NADPH oxidase, and liberating inflammatory cytokines and chemokines (Rodríguez-Pallares et al., 2008). These findings point to an involvement of APs in the physiopathology of PD. The decrease of APs activity may be responsible of autonomic and neuroendocrine dysfunctions. Potentially, APs would be used as biomarkers of PD and would provide information about progression of the disease.
We also isolated and characterized main plasma APs by electrophoretical techniques. Western blotting of denaturing AP allowed checking the existence of APs in plasma, although this technique was not suitable for enzymatic activity quantification. Also, results from non denaturing polyacrylamide gel electrophoresis (PAGE) revealed activity in the same direction (increase or decrease) to those obtained by fluorimetric assay, although it was not possible to quantify espectroscopically.
Furthermore, the LBs formation in dopaminergic neurons is related to the decreased protein degradation. ¿-Synuclein and ubiquitin are the main components in LBs (Cookson, 2005). The identification of these intracellular aggregates in living nigral neurons and the loss of dopaminergic neurons are both necessary to confirm the disease from the pathological point of view. Many studies have reported the detection of ¿-synuclein in biological fluids such as cerebrospinal fluid and plasma of PD patients (Borghi et al., 2000; El-Agnaf et al., 2003). It is a matter of debate if plasma ¿-synuclein is a protein secreted by normal neurons or released by damaged neurons. Our results showed significantly higher concentrations of plasma ¿-synuclein in both PD groups, PDt and PDnt respect to control group. Similar results were reported by El-Agnaf et al., (2006) and Lee et al., (2006). However, so far, this is the first paper reporting changes in ¿-synuclein in early diagnosed patients without treatment. The increased concentration of ¿-synuclein found in PDt and PDnt suggest that LBs are associated to neurodegeneration and that this is an early event in PD.
We analyzed statistically the possible correlation between plasma AP activities and ¿-synuclein levels, but data did not show any correlation. That is, the release of ¿-synuclein and aminopeptidases are independent, but increased levels of APs in plasma of PD patients would suggest an attempt of the organism to metabolize higher concentrations of circulating ¿-synuclein fragments.
We also analyzed changes in AP activity in other neurodegenerative disorders such as HD and AD to assess the specificity of changes in APs activity. We found a general decrease in AP activity in HD. This suggested that the decrease may be related to a progressive neuronal death in striatum, and it is probably also due to hypothalamic and neuroendocrine changes showed by HD patients (Petersén et al., 2006). Other HD feature is huntingtin (htt) aggregates. Many studies in vitro have shown that htt toxicity is occasioned by small fragments proceeding from the N-terminal degradation in the proteasome (Hook, 2006). Buthani et al. (2007) identified the cytosolic AlaAP as the main enzyme able to degrade these poli-Q sequences. It is possible that the decrease in the AP activity may be related to the characteristic proteolytic stress in HD, collaborating with htt toxicity by decreasing its metabolism.
Although APs activities in AD patients tended to increase, we did not find significant changes compared to controls. These data were different to those obtained in PDt and HD groups. Nevertheless, the time course of AD, the different treatments, and the remarkable aging must be considered and analyzed (there were significant differences between C and AD groups in age; P < 0.01). Statistical analysis showed a positive relationship between Ala- and GluAP activities and age (P < 0.01; P < 0.05; respectively). Thus, this factor could be responsible of the higher AP activity found in AD. As in PD and HD, the formation of protein aggregates is other pathologic feature of AD, i.e. Aß peptide and protein tau. Different studies reported a high heterogeneity in the N-terminal ends of Aß peptide (Saido et al., 1995; Saido en al., 1996), suggesting APs as key enzymes in its degradation. Kuda et al. (1997) reported a lower GluAP activity in plasma from AD patients, founding an opposite relation between Aß42 peptide concentration and GluAP activity in brain cortex of senile patients without dementia. Besides Aß peptide, protein tau was proposed as a substrate for APs. Segunpta et al. (2006) showed tau protein degradation in vitro by cytosolic AlaAP. Also, a higher expression of cytosolic AlaAP was reported in transgenic mice cerebellum expressing human mutant tau and the lower expression regions of AlaAP coincided with higher aggregation of tau protein. In addition, these data were observed in brain cortex of AD and frontotemoral dementia (FTD) patients. This suggests that the higher expression of AlaAP in specific regions may be a compensatory mechanisms adopted by living neurons after a long time progression to reduce tau-induced neurodegeneration (Karsten et al., 2006). In summary, APs activity may have a crucial role in brain metabolism of pathological proteins.
We also assayed AP activity in patients with a non degenerative, acute, neurological disease such as ischemic ictus (Ic). Our results showed that AlaAP, LeuAP, and GluAP activities did not change in Ic group compared to C group. However, CysAP and AspAP activities were lower in Ic group than in C group. Therefore, changes in the pattern of AP activity are different to those observed in neurodegenerative diseases. These differences on AP activity may be due to the localization, extension of damaged regions and the duration of ischemic ictus. These factors are different to neurodegenerative diseases, with similar affected regions in each group and being both chronic and progressive diseases.
The second main objective was to analyze the relationship between oxidative stress and neurodegenerative diseases. Focusing on PD, in a previous work we found higher endogenous plasma LPO concentrations in PD patients under treatment, suggesting that they are chronically under systemic oxidative stress (Agil et al., 2006). These findings are in agreement with present data that show significantly higher plasma LPO concentrations in PDt (see figure 10A). Moreover, we assayed plasma LPO concentration in de novo patients, PDnt group, and we found significantly higher LPO concentration in this group. However, there were not significant differences between both PD groups. These results suggest that although medication was able to normalize APs activities, it did not improve oxidative stress. Dexter et al. (1994) reported a lower reduced glutathione concentration in SN of asymptomatic PD patients than in healthy subjects, proposing that oxidative stress phenomena are previous to neuronal loss. Furthermore, we analyzed plasma LA concentration as other index of mitochondrial dysfunction. Data showed significantly higher plasma LA concentration in PDt. These data, together with those obtained in measure of LPO, suggest a direct relationship between mitochondrial dysfunction and free radicals production. Also, the fact that antiparkinsonian medication does not improve oxidative stress suggests that oxidative processes pursue damaging basal ganglia, and thus, despite treatment PD is progressing.
Plasma LPO and LA concentrations were analyzed in the others neurodegenerative diseases. Significantly higher plasma LPO concentration was found in HD group. Impaired metabolism and oxidative stress have been demonstrated in brain tissue for human and rodent model of HD (Browne et al., 1999). Although impaired metabolism and mitochondrial abnormalities have also been found in peripheral tissues, direct evidence of increased oxidative stress was rarely studied in peripheral blood of HD patients. Our findings are in agreement with other studies in HD, showing increases in plasma oxidative stress measured as malondialdehyde (MDA) (Chen et al., 2007), LPO and GSH (Klepac et al.,2007). Moreover, these studies reported a significant positive correlation between plasma MDA or LPO and disease severity, suggesting that an increase in oxidative stress takes place before clinical symptoms of HD. On the other hand, plasma LPO concentration showed an important decrease in AD. These data are the opposite of those obtained in previous studies, showing that oxidative stress phenomena are increased in AD (Andersen, 2004; Butterfield et al., 2007). These works suggest that oxidative stress is an early event in AD (Galbusera et al., 2004) and compensatory responses such as induction anti-oxidant enzymes, tau phosphorylation and NFT formation are activated in response to ROS production. Thus, the decrease in LPO concentration may be a compensatory response, and may provide some protective mechanisms to ensure neuronal survival (Zhu et al., 2004). Other alternative explanation is that the low LPO concentration found in AD group was a consequence of the long time course AD, and the activation of the compensatory responses or the prominent neuronal death produced a decrease in oxidative stress. It will be necessary to include a higher number of patients and to do an exhaustive analysis of plasma LPO concentration among AD patients to reach definite conclusions in this issue.
LPO and AL concentrations were also analyzed in Ic group. Our data showed a significant increase of LPO and AL concentrations in Ic. These findings are in agreement with the pathology of the disease, in which a complex cascade of metabolic events is initiated after brain ischemia and conclude with the ROS generation and reduced ATP production (Love, 1999; Martin et a., 2005). It is remarkable that in an acute disease as Ic, plasma LPO was elevated whereas in a long lasting progressive neurodegenerative disease as is AD, this oxidative stress marker was lower than in the control group.
In conclusion, APs activities change significantly in PD and others neurodegenerative diseases. Furthermore, specific treatment of PD leads not only to an improvement in motor symptoms, but also to normalization in plasma activities of APs. The others diseases showed a different pattern, with a tendency to an increase in AD, a decrease in HD, and a fluctuating pattern in the other group used as control of degenerative disease, Ic. Oxidative stress increase is a common feature of all neurodegenerative diseases as well as Ic, with the only exception of AD. If we take PD as a model, effective treatment of HD and AD must normalize APs activities and we believe that antioxidant protection treatment would be beneficial in all neurodegenerative diseases.
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