Biochemical analysis of protein stability in human brain collected at different post-mortem intervals.
Methods: Different neuroanatomical areas including frontal cortex (FC), cerebellum (CB), caudate nucleus (CD) and substantia nigra (SN) from autopsy human brains (n=9) with varying PMI (4-18 h) were analyzed for pH, protein insolubility, protein oxidation/nitration and protein expression of glial fibrillary acidic protein (GFAP), synatophysin and neurofilament (NF). Histological changes at different PMI were also assessed.
Results: An increase in tissue pH was noted with increasing PMI. Although there was no significant alteration in solubility of proteins, SN showed increased protein oxidation/nitration events, GFAP and NF expression with increasing PMI. No major abnormalities in cell morphology or tissue integrity were noted. Immunohistochemistry with GFAP and NF did not show any significant increase in signal in FC at high PMI.
Interpretation & conclusions: In post-mortem human brains, although there were no gross structural changes at the tissue level with increasing PMI, biochemical events such as oxidative and nitrosative damage of cellular proteins, tissue pH could be considered as markers of tissue quality for biochemical research. Further, SN was found to be most susceptible to PMI related changes.
Key words Glial fibrillary acidic protein--histology--human brain--neurofilament--3-nitrotyrosine--post-mortem interval--protein oxidation--tissue pH--Western blot
Increase in geriatric population in the society associated with pathophysiological changes in the nervous system and neurodegenerative disorders has necessitated detailed studies of pathogenesis to evolve therapeutic strategies. Though animal experimentation has allowed major advances in understanding neurological disorders, it has become clear that extrapolation to human system is not realistic because of species barrier, diversity in anatomy, physiology, biochemistry and genetics, except for some phenomenological similarities. Therefore, medical centers have initiated "brain banks" to collect and store human nervous tissue and body fluids for research (1). Human Brain Tissue Repository for Neurobiological Studies (HBTR-Human Brain Bank) has been established in 1995 in India at the department of Neuropathology, National Institute of Mental Health and Neuro Sciences (NIMHANS), Bangalore, as a national research facility to collect human nervous tissue and tissue fluids and provide to researchers (1). Such brain banks subserve the function of evolving a database on standardized samples and also to carry out "question-directed" studies to find clinically translatable answers. The use of brain tissue collected at autopsy for proteomic study of human neurobiology has increased with the Human Proteome Organization-Brain Proteome Project (HUPO-BPP) an international consortium aimed to promote exchanging raw data between groups and co-ordinate proteomic activities using the human brain (2,3).
But for successful application of proteomic technologies, the biochemical, molecular and structural integrity of the tissue should be well sustained. Pre-mortem factors like metabolic state of the deceased, infections, seizures, hypoxia, and consumption of toxic substances/drugs, terminal agonal state after death, post-mortem delay in extracting the brain, storage environmental temperature form critical modulators. Traditionally, a low post-mortem interval (PMI) has been a feature of high tissue quality and reliability of human brain (4-8). Recently, tissue pH and RNA quality have also been introduced as quality markers (9). Effect of post-mortem delay is important while studying post-translational modifications in human degenerative diseases. Proteomic studies of brain proteins to evaluate post-mortem changes have been carried out in animal models (10) but similar studies in human brain are scarce.
To facilitate detailed proteomic studies and to validate the nature of the brain tissue stored at HBTR, the present study was carried out, to evaluate the effect of PMI (the time interval between the time of death to the time the brain is collected at autopsy, sliced and stored at -70[degrees]C in a freezer) on the biochemical events in the brain.
Material & Methods
All the chemicals and solvents used were of analytical grade. Bulk chemicals and solvents were obtained from Merck & Co. Inc (Whitehouse Station, NJ, USA). Anti-neurofilament (phosphorylated H and M) mouse monoclonal antibody (clone NE-14), anti-glial fibrillary acidic protein (GFAP) mouse monoclonal antibody (clone GA-5), anti-synaptophysin mouse monoclonal antibody (clone Snp88) were obtained from Biogenex (San Ramon, CA, USA). Nitrocellulose membrane was obtained from Millipore (Billerica, MA, USA). Horse radish peroxidase conjugated secondary antibodies were obtained from Bangalore Genei (Bangalore, Kamataka, India). Anti-nitrotyrosine antibody (NT), anti-dinitrophenyl (DNP) antibody and protease inhibitor cocktail were procured from Sigma (Eugene, OR, USA).
Tissue samples: Tissue samples were collected from subjects between 25-35 yr to exclude the effect of age on biochemical parameters studied. Within 1 h of death, the body was transferred to a refrigerator maintained at 2-4[degrees]C with a recorder with uninterrupted power supply. Following medico-legal autopsy with inquest from police, with no prejudice to the medico-legal evaluation, brains were collected, with written informed consent from next of kin to use the material for research/teaching purposes. The Institutional Scientific Ethics Committee has approved the study protocol. The victims had no history of neurological disorder prior to death, as indicated in the records. The brains were sliced coronally and kept flat on salt-ice mixture (-15 to -18[degrees]C) during dissection and then transferred in plastic zip lock bags into a box to be stored at -80[degrees]C in HBTR. The procedure of dissection took 3045 min and the brain slices were transferred immediately into the deep freezer. Frozen brain samples from nine subjects (age 30 [+ or -] 5 yr, non-alcoholics, non-diabetics, not on any medication) with no visible signs of brain damage, from four anatomical areas: frontal cortex (superior frontal gyrus) (FC), cerebellum (CB), caudate nucleus (head) (CD) and substantial nigra (linear darkstrip above crus cerebri, both sides at the level of superior colliculus) (SN) were collected for biochemical study. The mirror image bits fixed in buffered formalin were processed for histological evaluation.
Preparation of protein extracts: Approximately 100 mg of frozen tissue from each sample was dissected and immediately transferred to cold 1x phosphate buffered saline (PBS) containing protease inhibitor cocktail (Sigma, Eugene, OR, USA) and manually homogenized on ice (15 strokes). The homogenate was sonicated (20 see on ice) in a Sonics-vibra cell sonicator (Sonics and Materials Inc, CT, USA). Total protein in the sonicated samples was estimated by Bradford method (11), aliquoted and stored at -80[degrees]C until further processing.
For preparation of triton-soluble extracts, the sonicate from each sample was diluted with 1x PBS containing protease inhibitor and Triton X-100 (Loba Chemie, Mumbai, India) (1% final concentration) up to 200 [micro]l at 5 [micro]g/[micro]l protein concentration and mixed thoroughly for complete solubilization (12). The solubilized material was centrifuged at 15,000 g (15 min, 4[degrees]C) and 10 [micro]l of supernatant (Triton-soluble fraction) was loaded onto 12 per cent SDS PAGE. Similarly, the pellet (Triton-insoluble fraction) was solubilized in 10 [micro]l SDS PAGE loading dye, boiled and loaded onto a 12 per cent SDS PAGE followed by staining with coomassie brilliant blue R-250 (Sigma, USA). From corresponding soluble and insoluble fractions, band intensities were quantified by a densitometric scanner (Bio-Rad laboratories, Hercules, CA, USA) and (insoluble fraction)/(insoluble + soluble fraction) ratio, which is a gross indicator of the level of insoluble proteins, was calculated.
SDS PAGE and Western blot: Brain total protein extracts (approximately 50 [micro]g/lane) were run on 12 per cent SDS PAGE at 100 V for about 2.5 h and gels were stained with coomassie brilliant blue R-250 (13). For Western blot, proteins from SDS gels were electrophoretically transferred to nitrocellulose membranes in a semi-dry apparatus (Sree Maruthi Scientific Works, Bangalore, India) (2 h at 125 mA) (13). Non-specific binding was blocked by incubating membranes in 1x PBS containing Tween-20 and 5 per cent skimmed milk powder (Nandini Milk Products, Bangalore, India) (for 1 h at room temperature or overnight at 4[degrees]C). The membranes were incubated with primary antibodies diluted in PBS/Tween-20 containing 5 per cent BSA for 1.5 h at room temperature. Blots were washed with PBS/Tween-20 and then incubated for 1.5 h at room temperature with HRP-conjugated secondary antibodies (in PBS/Tween-20 containing 5% BSA). Membranes were washed with PBS/Tween-20 and the immune reaction was visualized by the colour reaction developed in 1x PBS containing diamino benzidine (DAB) [1mg/ml (w/v)] (Sigma) and 0.1 per cent [H.sub.2][O.sub.2] (13). Anti-tubulin (Calbiochem, San Diego, CA, USA) westerns were used as internal control.
To detect endogenous protein nitration, equal amounts of protein (100 [micro]g) from different brain samples were spotted in triplicate onto a nitrocellulose membrane. The membrane was washed with PBS/ Tween-20 followed by Western blot with polyclonal anti-3-NT antibody. Band intensities in westerns were quantified by a densitometric scanner and the values normalized against the respective anti-tubulin signal.
Estimation of protein carbonyls (oxyblot): Oxyblots were carried out based on the protocol published earlier (14). Brain protein extract prepared as indicated was centrifuged (14,000 g/ 15 min/4[degrees]C). The supernatant at 4 mg/ml protein concentration was derivatized by dinitrophenyl hydrazine (DNPH) (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) (in 50% sulphuric acid) in a final reaction volume of 20 [micro]l in the presence of 12 per cent SDS for 20 min at room temperature. The reaction was stopped by addition of neutralization buffer (2M Tris in 30% glycerol). Sample (5[micro]l) was spotted in triplicate onto a nitrocellulose membrane. The membrane was washed with PBS/Tween-20 followed by Western blot with polyclonal anti-DNP antibody.
pH determination: pH determination in brain tissue with FC as representative for each brain was carried out based on the method described earlier (9). Approximately 150 mg of frozen frontal cortex tissue from different subjects was homogenized in 5 ml of saline (pH=7.0) and centrifuged (3 min/8000 x g/4[degrees]C). The pH of the supernatant was measured in duplicate with a pH meter (Control Dynamics Instrumentation, Bangalore, India) that was previously calibrated with known standards.
Histological study: The paraffin embedded tissue sections from four different anatomical areas from all the subjects were stained with haematoxylin-eosin (HE), Nissl and Luxol fast blue for myelin. The sections were examined blind to post-mortem delay for myelin pallor, neuronal staining character and anoxic changes if any. The sections from lowest PMI (ID B-183) and longest PMI (ID T-144) were immunostained with antibodies to GFAP (Glial marker, 1:200) and neurofilament (axonal marker, 1:100) to note if there is any difference in staining character. Appropriate positive and negative controls were incorporated during immunostaining. The antigen retrieval was carried out by microwaving at level III, in citrate buffer followed by standard immunoperoxidase technique, with DAB/[H.sub.2][O.sub.2] as chromogen.
Statistical analyses: Quantitative data wherever mentioned were accumulated from at least three independent experiments. Differences between mean values were analyzed by one-way analysis of variance (ANOVA).
Biochemical analysis of FC, CB, CD and SN regions from nine human brain samples collected at autopsy with increasing PMI (4-18 h) (Table I) was carried out. The tissue pH in FC revealed an apparent increase with advanced PMI in collection of tissue (4 h PMI- pH 6.25 to 18 h PMI- 6.77) (Table II). Total proteins from Triton X-100 soluble and insoluble fractions when subjected to SDS-PAGE and quantitation of the profile showed no significant elevation of insoluble proteins with increasing PMI in all the four brain regions (Fig. 1).
Slot blot assay with anti-3-NT antibody as a marker of protein nitration and quantitation following normalization with anti-tubulin signal (Figs. 2&3) showed significant increase in nitration levels in SN, mild in CB and no alteration in FC and CD with increasing PMI. On similar lines, oxyblot of total protein carbonyls in protein extracts from human brain samples with increasing PMI showed an elevation in protein carbonyl levels as well in SN whereas there was no significant change in other regions (Fig. 4).
Western blot analysis of selected proteins in the brain such as glial fibrillary acidic protein (GFAP), synaptophysin and neurofilament (NF) at increasing PMI and quantitation (normalized against tubulin) showed no significant change in GFAP signal in FC, CB and CD, while SN revealed a significant increase in GFAP signal (Fig. 5). Quantitation of Western blot signal of synaptophysin, a synaptic vesicle glycoprotein present in virtually all neurons in the brain that participate in synaptic transmission showed no significant change in CB, CD and SN, albeit there was a mild increase in FC (Fig. 6). Neurofilaments (NFs) are intraneuronal and axonal cytoskeletal framework proteins. NF proteins on SDS PAGE separate into three polypepides based on increasing molecular weight (NF-L, Mol wt: 68-70 kDa; NF-M, Mol. Wt: 145-160 kDa and NF-H, Mol. wt: 200-220 kDa). With increasing maturity of the human brain, NF-L levels recede while the levels of NF-M and NF-H become apparent. In this study on adult brains, Western blots could detect only the NF-M and NF-H species (Fig. 7). In some samples, lower molecular weight bands (~25 and 50 kDa), probably representing degraded peptides of NF were noted but not relating with PMI. Quantitation of the NF signal (normalized against tubulin signal) showed a significant (P<0.05) increase in SN and a decrease in FC, while it was marginal in CB and CD (Fig. 7). Histological examination of the sections from four anatomical areas at different times of post-mortem delay (9 samples) revealed essentially similar features of cytoarchitecture and density of myelin. The immunolabelling character for GFAP and NF (Fig. 8) was essentially similar at 4 and 18 h PMI.
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PMI is considered to influence the tissue quality for biochemical and molecular biological studies on human brain tissue samples. Stan et al (9) analyzed post-mortem brain tissue collected at fixed PMI (16-20 h) but wide age range (15-90 yr) from control subjects (succumbed to sudden cardiac causes with short agonal events but no neurological disorders) and from individuals with neurological disorders. Their study suggested that the most useful indicator of good tissue quality is RNA integrity and brain tissue pH. Further, even when RNA was found degraded, the protein levels remained stable in their samples. Similar studies (15-17) indicate that human brain collected at autopsy, if stored appropriately yields high quality RNA suitable for molecular biological and proteomic studies.
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The different anatomical areas of brain depending on specialized physiological functions and neuronal connections have varied cellular characters and neurotransmitter profiles. Hence the present study was aimed at analyzing the biochemical and histological changes at four anatomical brain regions: frontal cortex (FC)-cholinergic, cerebellum (CB)- GABAergic, head of caudate nucleus (CD) and substantia nigra (SN)-dopaminergic, with increasing PMI (4-18 h) within a close age range of subjects (30 [+ or -] 5 yr) ensuring the bodies after death were subjected to identical (very narrow range) post-mortem handling and preservation of the tissue in the brain bank. The tissue pH, solubility of proteins, oxidation and nitration status (markers of post-translational modification) and relative stability of proteins like GFAP, NF (relatively stable structural proteins) and synaptophysin (a synaptic vesicle glycoprotein) were used as markers of biochemical changes in these regions, with increasing PMI.
Brain tissue pH is considered to reflect postmortem tissue integrity. Within any brain region, due to their divergent physiological activity and resultant metabolic rate, the native neuronal proteins could be susceptible to factors that affect the local pH and tissue quality. The pH measurement in the FC (not estimated in other areas due to limitation of precious human post-mortem material) indicated a marginal elevation in increasing PMI, as reported earlier (9).
Protein aggregation and insolubility that occur with normal ageing and neurodegenerative diseases reflect altered protein function (18). Earlier we reported increased protein aggregation and formation of neurofibrillary tangles rich in hyperphosphorylated neurofilament tau and ubiquitin and amyloid [beta] protein plaques of varied maturity in non-demented individuals (control) beyond 60 yr of age and further higher density in Alzheimer's disease (AD) in the frontal cortex and hippocampus (19). These areas also reflected enhanced labelling for GFAP indicating gliosis, but relatively stable synaptophysin staining in normal controls and depletion of synaptophysin in cases of AD (unpublished observations). The estimation of insoluble proteins in our samples showed no significant difference in different anatomical areas studied. This indicates absence of enhanced protein aggregation during early post-mortem events and relative stability for proteomic studies.
Nitrosative stress mediated by proxinitrite (PN, [ONOO.sup.-]) and nitric oxide (N[O.sup.-]) are known to cause cellular changes in vivo. PN, a short-lived highly reactive nitrogen species (RNS) generated in mitochondria rapidly damages proteins by post-translational modifications. PN mediated 3-NT modification may be significant for cellular damage during physiological aging (20) and neurodegenerative disorders like AD and Parkinson's disease (PD) (21-22) and during mitochondrial dysfunction (23-25). PN mediated nitration and nitrosation events have been shown to inhibit mitochondrial complex I enzyme, an event relevant to pathogenesis of PD (26) suggesting increased nitration of cellular proteins could be a marker of cellular pathophysiology. Significantly enhanced 3-NT levels mainly in SN unlike other areas were noticed which could be a secondary effect due to the localization of dopaminergic system and neuromelanin that makes SN vulnerable to oxidative and nitrosative stress (27).
During normal physiological ageing and neurodegeneration, reactive oxygen species dependent direct oxidation of side chains of amino acids such as cys, arg, pro, thr, etc., occurs, altering the structure-function relationship of proteins (28) in cells thus reflecting pathophysiological events. Fetter et al observed no evidence of protein degradation in the human brain samples stored at 1[degrees]C and then frozen for different intervals up to 50 h post-mortem (5). However, the levels of several proteins were modified in samples stored at 4[degrees]C and furthermore when stored at room temperature. However, the levels of several proteins were modified in samples stored at 4[degrees]C and further more when stored at room temperature. Hence they emphasized the need for reducing the body temperature after death to minimize protein modification and degradation. In the present study, enhanced protein carbonyl could not be detected in the regions studied.
Following stress, infection and injury to the nervous system, a reparative astrocyte proliferation and reactive change is initiated within 24 h with enhanced expression of the astrocyte specific fibrous protein, GFAP (29,30). Significant correlation between age and number of GFAP positive astrocytes in hippocampus in human brains have been reported, but no correlation has been reported between GFAP expressing astrocytes, the cause of death and post-mortem delay in collecting brain samples (31). The marginal increase in GFAP expression in SN in our study in contrast to other zones could be related to neuroanatomical variation than the post-mortem delay. On the other hand, markers like synaptophysin showed no significant change in expression levels in Western blots, though immunohistochemical labelling was of low intensity. The expression pattern of neurofilament peptides and the presence of truncated NF on blots with no specific association to PMI delay indicate early susceptibility of this neuro-axonal intermediate filament to phosphatases and proteases and post-translational modifications. The enhanced signal for NF in SN probably reflects the regional difference in the presence of dense axonal bundles traversing it, in comparison to FC, CB and CD.
The biochemical data were viewed with due consideration to the clinical history and the type of injury causing the death. Samples B-183, B-182, T-150 and T-144 were derived from subjects who succumbed to spinal injury and the brain was normal. The others were samples from individuals who sustained head trauma, though the regions used for the study were normal and anatomically away from the site of injury. Careful examination of the data of these two sub groups did not indicate any direct influence of tissue injury on the biochemical parameters in the brain sampled.
Histopathology showed that at the tissue level there were no major abnormalities in cell morphology or tissue integrity with increasing PMI (data not shown). There was a small increase in GFAP levels that were corroborated with the westerns probably related to ante-mortem brain response to trauma as a reparative phenomenon. Overall, our analysis showed that SN region was more prone to oxidative, nitrative and other biochemical changes with increasing PMI, which is probably related to the dopaminergic profile of the area. As suggested earlier (9), before autopsy, when the bodies are maintained at 4[degrees]C, the inner regions of the brain cool more slowly than the surface. Therefore, it is possible that the brain tissue in the deep areas such as CD and SN could be subject to some of the oxidative and hydrolytic enzyme reactions due to differential cooling, altering the integrity of some of the proteins. It is essential to keep in mind these limitatiolas in interpreting the biochemical and proteomic profile in brain tissue collected at autopsy. Studies on human brain are essential for extrapolating from animal and in vitro studies due to differences in biochemical pathways, species barrier, etc., and the need to understand in a more natural environment. More studies on human autopsy brains obtained beyond 18 h PMI are required to understand and confirm the occurrence of such biochemical effects during larger PMI. Further studies to understand the role of these protein markers in human post-mortem brains during normal brain ageing and during neurodegenerative diseases such as PD are required.
This work was supported by an International research grant from Parkinson's Disease Foundation, USA and a Fast Track Grant from DST, India both to M.M.S.B. R.M is supported by a junior research fellowship from CSIR, India. We gratefully acknowledge the Human Brain Tissue Repository (HBTR) for Neurobiological Studies (Human Brain Bank), Department of Neuropathology, NIMHANS, Bangalore, for providing human brain tissue samples. The authors would like to thank Dr Anu Rangarajan, Indian Institute of Science, Bangalore, India for the kind gift of anti-tubulin antibody. This study is dedicated to the memory of Prof. Taranath Shetty, Professor of Neurochemistry, NIMHANS.
Received November 21, 2007
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Reprint requests: Dr M.M. Srinivas Bharath, Assistant Professor, Department of Neurochemistry, National Institute of Mental Health & Neuro Sciences (NIMHANS), EB # 2900, Hosur Road, Bangalore 560 029, India e-mail: bharath@ nimhans.kar.nic.in
Ramesh Chandana, R.B. Mythri, Anita Mahadevan *, S.K. Shankar * & M.M. Srinivas Bharath
Departments of Neurochemistry & * Neuropathology, National Institute of Mental Health & Neuro Sciences Bangalore, India
Table 1. Details of brain samples with increasing PMI collected from road traffic accident victims-Human Brain Bank (n=9: average age 3 [+ or -] 5 yr) Sample ID Age Interval (yr)/sex between head injury to death B-183 27/F 2 h 45 min T-149 25/M 12 h 30 min B-180 25/M 13 h B-178 35/M 30 h 15 min T-146 33/M 11 h B-182 25/M 17 h 30 min T-150 30/M 3 h T-148 35/M 8 h T-144 25/M 27 h 45 min Sample ID Interval Total from time interval of death from injury to to freezing freezing brain (post brain at mortem -80[degrees]C delay) B-183 4 h 6 h 45 min T-149 7 h 30 min 20 h B-180 7 h 30 min 20 h 30 min B-178 12 h 42 h 15 min T-146 14 h 30 min 25 h 30 min B-182 14 h 30 min 32 h T-150 16 h 30 min 19 h 30 min T-148 17 h 25 h T-144 18 h 45 h 45 min Sample ID Type of injury Regions studied B-183 Poly trauma, no brain injury FC, CB, CD T-149 SDH, Lt parietal, Rt. frontal FC, CB, CD, SN contusion B-180 Acute SDH-evacuated cerebral FC, CB, CD oedema. CT-brainstem hypodensity B-178 Lt. Temporo-parietal EDH-drained FC. CB, CD cerebral oedema T-146 Acute SDH-evacuated Lt fronto FC, CB, CD, SN temporal contusion, cerebral oedema B-182 High cervical cord injury-sudden FC, CB, CD, SN cardiac arrest. Brain normal T-150 Medullary tear. Normal brain FC, CB, CD, SN T-148 Right fronto- temporal SDH. FC, CB, CD, SN cerebral oedema T-144 Cervical cord injury. Brain normal, FC, CB, CD, SN All deaths occurred during January-April 2007, between 18:45 - 05:30 h. Ambient temp. 10-22[degrees]C. Body shifted to mortuary freezer within 60 min after death. Mortuary freezer maintained at 2-4[degrees]C with temperature recording. At emergency service for the injury victim oxygen saturation was maintained by intubation and oxygen mask. All received only antioedema measure fluid correction and terminally one vial of adrenaline, atropine and steroid when indicated. None of the victims were alcoholics/diabetic/not on any medication. SDH, subdural haematoma; EDH, extradural haematoma; FC, frontal cortex; CB, cerebellum; CD, caudate nucleus; SN, substantia nigra. Table II. pH estimation in frontal cortex obtained across different post-montem interval (PMI) Sample no. PMI (h) pH value [+ or -] 0.1 (SD B-183 4 6.25 T-149 7.5 6.22 B-180 7.5 6.42 B-178 12 6.01 T-146 14.5 6.30 B-182 14.5 6.56 T-148 17 6.82 T-144 18 6.77
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|Author:||Chandana, Ramesh; Mythri, R.B.; Mahadevan, Anita; Shankar, S.K.; Bharath, M.M. Srinivas|
|Publication:||Indian Journal of Medical Research|
|Date:||Feb 1, 2009|
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