Characterization of Postmortem Human Brain Proteins by Two-Dimensional Gel Electrophoresis

Abstract: The proteins of membrane and cytosol fractions from frozen human postmortem brain were analyzed by two-dimensional gel electrophoresis (isoelectric range: 5.1–6.0) and both Coomassie-blue and ammoniacal silver staining. Cytosol preparations were analyzed from six different postmortem brains from patients with various neurologic diagnoses and immediate causes of death. Intervals between death and brain freezing (−70^oC) ranged from 2 to 20 h. The vast majority of proteins detected in these cytosol fractions had identical molecular weights and isoelectric points in each of six human brains examined. However, in some tissue samples tubulin was either quantitatively decreased or undetectable. The possibility that this partial or complete depletion of tubulin was related to postmortem interval and/or brain freezing was studied using rat forebrain tissue. Rat brain incubated at room temperature for up to 24 h did not reproduce the changes seen in the region of human cytosol tubulin. However, other changes seen in the two-dimensional electrophoretic pattern of rat cytosol proteins did relate to postmortem interval, brain freezing, or both. Rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum were prepared from three human brains, with highly reproducible two-dimensional patterns. Protein analysis of these membrane fractions revealed that human RER contained significant amounts of tubulin, in contrast to rat RER which contained no detectable tubulin. This discrepancy was elucidated by allowing rat brains to remain at room temperature for 24 h before freezing; gels of rat RER prepared from this tissue showed that tubulin subunits were present.     

Pre-and Postmortem Influences on Brain RNA

Abstract: Many potentially valuable techniques for the understanding of human neurobiological and neuropathological processes require the use of RNA obtained from postmortem tissue. As with earlier neurochemical studies, there are two particular problems posed by such tissue in comparison with tissue from experimental animals. These are the postmortem interval and the condition of the patient prior to death, referred to as the agonal state. We review the nature and extent of the effects of postmortem interval and agonal state on RNA in brain tissue, with particular reference to the study of neuropsychiatric disorders. Perhaps surprisingly, postmortem interval has at most a modest effect on RNA. Abundant intact and biologically active RNA is present in tissue frozen 36 h or more after death. Postmortem interval does not account for the marked variability observed among human brains in all RNA parameters. Despite the overall stability of RNA after death, some evidence suggests that individual RNAs may undergo postmortem decay. Less attention has been paid to the effects of agonal state. The existing data indicate that events in the premortem period such as hypoxia and coma can affect the amount of some messenger RNAs. The nature of agonal state influences depends on the messenger RNA in question, though the basis for this selective vulnerability is unknown. No agonal state effect on overall RNA level or activity has been found. The data show that postmortem brain tissue can be used for RNA research. However, considerable attention must be paid to controlling for the influences of pre-and postmortem factors, especially when quantitative analyses are performed.     

The postmortal accumulation of brain N-arachidonylethanolamine (anandamide) is dependent upon fatty acid amide hydrolase activity

N-arachidonylethanolamine (AEA) accumulates during brain injury and postmortem. Because fatty acid amide hydrolase (FAAH) regulates brain AEA content, the purpose of this study was to determine its role in the postmortal accumulation of AEA using FAAH null mice. As expected, AEA content in immediately frozen brain tissue was significantly greater in FAAH-deficient (FAAH-/-) than in wild-type mice. However, AEA content was significantly lower in brains from FAAH-/- mice at 5 and 24 h postmortem. Similarly, wild-type mice treated in vivo with a FAAH inhibitor (URB532) had significantly lower brain AEA content 24 h postmortem compared with controls. These data indicate that FAAH contributes significantly to the postmortal accumulation of AEA. In contrast, the accumulations of two other N-acylethanolamines, N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), were not reduced at 24 h postmortem in either the FAAH-/- mice or mice treated with URB532. FAAH-/- mice accumulated significantly less ethanolamine at 24 h postmortem compared with wild-type mice, suggesting that FAAH activity plays a role in the accumulation of ethanolamine postmortem. These data demonstrate that FAAH activity differentially affects AEA and OEA/PEA contents postmortem and suggest that AEA formation specifically occurs via an ethanolamine-dependent route postmortem.     

CYCLIC NUCLEOTIDES and PROTEIN KINASE ACTIVITY IN THE RAT BRAIN POSTMORTEM

Abstract— The components of the cyclic nucleotide system were studied in rat brain at various times after death to determine the stability of the system postmortem. The concentration of cyclic AMP in 4 brain regions was highest 10 min after death and declined to stable levels within 6 h after death. At this time concentrations approximated those normally seen in vitro. The concentration of cyclic GMP in brain regions fell markedly postmortem and was undetectable 6 or 16 h after death. Nor-epinephrine-stimulated cyclic AMP accumulation measured in vitro in slices of the cerebral cortex was the same 10 min and 16 h postmortem. In the cerebellum, however, accumulated levels of cyclic AMP were lower 16 h postmortem, although the degree of stimulation elicited by norepinephrine was the same 10 min and 16 h after death. Cyclic AMP-dependent and independent protein kinase activities were detected in the rat brain at all times after death. There was no change in the cyclic AMP-dependent enzyme activity postmortem, but activity in the absence of cyclic AMP was significantly higher immediately after death compared to 6 or 16 h postmortem. These studies indicate that it should be possible to study the cyclic AMP system in human brain tissue obtained at autopsy.     

Methyl- and acetyltransferases are stable epigenetic markers postmortem

Postmortem brain tissue has been reported to be suitable to delineate regional pattern of possible disturbances underlying epigenetic functionality. However, from many parameters that have been detected in postmortem brain regions it is noteworthy that an effect of postmortem interval (PMI), storage time and premortem parameters should not be underestimated. Our previous investigation revealed that tryptophan (TRP) levels in postmortem brain tissue is affected by PMI and storage time. Since, alteration in TRP levels are assumed to be due to protein degradation, we further investigated whether TRP correlates to variables such as RNA, proteins and DNA modulators. In addition, we aimed to elucidate whether established postmortem variables may influence epigenetic parameters. These were investigated in well characterized postmortem human brain tissue originating from the European Brain Bank consortium II (BNEII). We could confirm previous findings, in which some protein levels alter because of prolonged PMI. Similarly, we demonstrated an influence of increased storage period on TRP levels, which might indicate degradation of proteins. Still not all proteins degrade in a similar manner, therefore a specific analysis for the protein of interest would be recommended. We found that methyltransferase- and acetyltransferase-activities were relatively preserved with PMI and storage duration. In conclusion, preservation of acetyltransferase- and methyltransferase-activities provides possible evidence of stability for epigenetic studies using postmortem tissue.     

Depletion of glutathione does not affect electron transport chain complex activity in brain mitochondria: Implications for Parkinson disease and postmortem studies

Glutathione is an important antioxidant in the brain that appears to be decreased, in conjunction with mitochondrial complex I activity, in Parkinson disease patients. In postmortem analysis, measurement of glutathione levels and complex I activity can be delayed up to 20h. We investigated whether depletion of glutathione in the preweanling rat induces a reduction in complex I activity in brain mitochondria and the effects that postmortem delay has on glutathione levels and electron transport chain activity. After injection with the glutamate-cysteine ligase inhibitor, buthionine sulfoximine (L-BSO), glutathione levels were decreased by 53% compared to the control values in whole-brain homogenates. During postmortem delay of 24h, in which animals were kept at 4°C, the levels of glutathione decreased in the control group by 58% and in the L-BSO-treated group by 79%. However, during this period, there were no changes in mitochondrial electron transport chain complex I, II-III, or IV activity in either group. These results suggest that a preexisting deficiency of glutathione or a loss of glutathione during postmortem delay does not influence mitochondrial respiratory chain activity in the brain.     

Tryptophan is a marker of human postmortem brain tissue quality

Postmortem human brain tissue is widely used in neuroscience research, but use of tissue originating from different brain bank centers is considered inaccurate because of possible heterogeneity in sample quality. There is thus a need for well-characterized markers to assess the quality of postmortem brain tissue. Toward this aim, we determined tryptophan (TRP) concentrations, phosphofructokinase-1 and glutamate decarboxylase activities in 119 brain tissue samples. These neurochemical parameters were tested in samples from autopsied individuals, including control and pathological cases provided by 10 different brain bank centers. Parameters were assessed for correlation with agonal state, postmortem interval, age and gender, brain region, preservation and freezing methods, storage conditions and storage time, RNA integrity, and tissue pH value. TRP concentrations were elevated significantly ( p  = 0.045) with increased postmortem interval; which might indicate increased protein degradation. Therefore, TRP concentration might be one useful and convenient marker for estimating the quality of human postmortem brain tissue.     

Differential in vitro metabolism of beta-endorphin in schizophrenia

Biologically active peptide fragments derived from the proteolytic cleavage of beta-endorphin (beta E) have been shown to be present in the brain. Based on clinical results using some of these fragments in neuropsychiatric disease studies we investigated the in vitro metabolism of beta E by twice-washed membrane homogenates of postmortem putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that frozen (-80 degrees C) postmortem human tissues are viable for these studies and that metabolism in control tissue proceeds similarly to fresh tissues. Furthermore, a significant increase in the formation of the putative neuroleptic-like peptide fragment des-enkephalin-gamma-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of beta E was also reported. These data suggest that an approach using postmortem human brain is possible in studying beta-endorphin catabolism and is therefore applicable to other neuropeptide systems.     

Brain Banks Spur New Frontiers in Neuropsychiatric Research and Strategies for Analysis and Validation

Neuropsychiatric disorders affect hundreds of millions of patients and families worldwide. To decode the molecular framework of these diseases, many studies use human postmortem brain samples. These studies reveal brain-specific genetic and epigenetic patterns via high-throughput sequencing technologies. Identifying best practices for the collection of postmortem brain samples, analyzing such large amounts of sequencing data, and interpreting these results are critical to advance neuropsychiatry. We provide an overview of human brain banks worldwide, including progress in China, highlighting some well-known projects using human postmortem brain samples to understand molecular regulation in both normal brains and those with neuropsychiatric disorders. Finally, we discuss future research strategies, as well as state-of-the-art statistical and experimental methods that are drawn upon brain bank resources to improve our understanding of the agents of neuropsychiatric disorders.     

Dynamic changes of the phosphoproteome in postmortem mouse brains

Protein phosphorylation is deeply involved in the pathological mechanism of various neurodegenerative disorders. However, in human pathological samples, phosphorylation can be modified during preservation by postmortem factors such as time and temperature. Postmortem changes may also differ among proteins. Unfortunately, there is no comprehensive database that could support the analysis of protein phosphorylation in human brain samples from the standpoint of postmortem changes. As a first step toward addressing the issue, we performed phosphoproteome analysis with brain tissue dissected from mouse bodies preserved under different conditions. Quantitative whole proteome mass analysis showed surprisingly diverse postmortem changes in phosphoproteins that were dependent on temperature, time and protein species. Twelve hrs postmortem was a critical time point for preservation at room temperature. At 4°C, after the body was cooled down, most phosphoproteins were stable for 72 hrs. At either temperature, increase greater than 2-fold was exceptional during this interval. We found several standard proteins by which we can calculate the postmortem time at room temperature. The information obtained in this study will be indispensable for evaluating experimental data with human as well as mouse brain samples.     

Agonist-Induced, GTP-Dependent Phosphoinositide Hydrolysis in Postmortem Human Brain Membranes

Abstract: Membranes prepared from postmortem human brain were used to measure the activities of three components of the phosphoinositide second messenger system. [³H] Phosphatidylinositol ([³H] PI) hydrolysis was stimulated by directly activating phospholipase C with calcium, by activating guanine nucleotide-binding proteins (G proteins) with guanosine-5′-O-(3-thiotriphosphate) (GTPγS) or with AIF₄, and by receptors activated with several agonists (in the presence of GTPγS), including (in order of increasing magnitudes of responses) carbachol, pilocarpine, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (a selective excitatory amino acid metabotropic receptor agonist), serotonin, and ATP. G_q/11 was identified as the G protein most likely to mediate [³H] PI hydrolysis in human brain membranes based on the findings that this process was not impaired by pretreatment with pertussis toxin and it was inhibited by antibodies specific for the α-subunit of G_q/11 but not by antibodies for G_o or G₁₁. The effects of postmortem delay on [³H] PI hydrolysis were examined by studying tissues obtained 6–21 h postmortem. A slight increase in basal [³H] PI hydrolysis was associated with increased postmortem time, suggesting a slow loss of the normal inhibitory control of phospholipase C. GTPγS- stimulated [³H] PI hydrolysis was unaffected by postmortem times within this range, but carbachol-induced [³H] PI hydrolysis tended to decrease with increasing postmortem times. These results demonstrate that the entire phosphoinositide complex remains functional and experimentally detectable in postmortem human brain membranes. This method provides a means to study the function, regulation, effects of diseases, and responses to drugs of the phosphoinositide system in human brain.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

Differences in postmortem stability of sex steroid receptor immunoreactivity in rat brain.

Difficulties in demonstrating sex steroid receptors in the human brain by immunohistochemistry (IHC) may depend on postmortem delay and a long fixation time. The effect of different postmortem times was therefore studied in rat brain kept in the skull at room temperature for 0, 6, or 24 hr after death. After a long fixation for 20 days, hypothalami were embedded in paraffin and sections were immunohistochemically stained for androgen receptor (AR), estrogen receptor-alpha (ER), or progesterone receptor (PR). Retrieving the antigenic sites by microwave pretreatment was essential to obtain successful IHC in all groups studied. In general, immunoreactivity was restricted to the cell nuclei. However, the intensity of the staining appeared to be strongly dependent on the different receptor antigens and postmortem time. Both AR and ER but not PR immunoreactivity were decreased after immersion-fixation compared to the perfused sections at time point zero. In brains fixed by immersion, all three receptors decreased gradually with increasing postmortem time, and ER became hardly detectable after 24 hr postmortem. The results of these experiments show that, with the protocol used, postmortem variables and lengthy fixation do not, in principle, prevent sex steroid receptor IHC in human material. The outcome of the immunostaining, however, might be strongly dependent on the epitopes and/or antibody used.     

Evidence of Glutamatergic Denervation and Possible Abnormal Metabolism in Alzheimer''s Disease

Excitatory dicarboxylic amino acids previously have been ascribed several functions in the brain. Here their total concentration and proposed neurochemical markers of neurotransmitter function have been measured in brain from patients with Alzheimer's disease (AD) and controls. Specimens were obtained antemortem (biopsy) approximately 3 years after emergence of symptoms and promptly (less than 3 h) postmortem some 10 years after onset. Early in the disease a slight elevation in aspartic acid concentration of cerebral cortex was observed in the patients with AD. A reduction in glutamic acid concentration of a similar magnitude was found. It is argued that this, together with a decrease in CSF glutamine content and lack of change in the phosphate-activated brain glutaminase activity of tissue, reflects an early metabolic abnormality. Later in the disease evidence of glutamatergic neurone loss is provided by the finding that in many regions of the cerebral cortex the Na+-dependent uptake of D-[3H]aspartic acid was almost always lowest in AD subjects compared with control when assessed by a method designed to minimise artifacts and epiphenomena. Release of endogenous neurotransmitters from human brain tissue postmortem did not appear to have the characteristics of that from human tissue antemortem and rat brain.     

Relationship between tryptophan and serotonin concentrations in postmortem human brain

The present study was planned to test a recent observation of positive correlation between tryptophan and 5-hydroxyindole concentrations in postmortem human hypothalamic samples. Four other brain areas were studied, but no significant correlations were observed between tryptophan and serotonin or 5-hydroxyindoleacetic acid concentrations, except in the nucleus accumbens samples of a suicide victim group. A possible in vivo correlation may have been obscured by postmortem changes. The use of tryptophan concentrations as an index for normalising postmortem brain serotonin data is not supported by the present results.     

Retracted: Aging is associated with altered inflammatory,arachidonic acid cascade,and synaptic markers,influenced by epigenetic modifications,in the human frontal cortex

Aging is a risk factor for Alzheimer's disease (AD) and is associated with cognitive decline. However, underlying molecular mechanisms of brain aging are not clear. Recent studies suggest epigenetic influences on gene expression in AD, as DNA methylation levels influence protein and mRNA expression in postmortem AD brain. We hypothesized that some of these changes occur with normal aging. To test this hypothesis, we measured markers of the arachidonic acid (AA) cascade, neuroinflammation, pro‐ and anti‐apoptosis factors, and gene specific epigenetic modifications in postmortem frontal cortex from nine middle‐aged [41 ± 1 (SEM) years] and 10 aged subjects (70 ± 3 years). The aged compared with middle‐aged brain showed elevated levels of neuroinflammatory and AA cascade markers, altered pro and anti‐apoptosis factors and loss of synaptophysin. Some of these changes correlated with promoter hypermethylation of brain derived neurotrophic factor (BDNF), cyclic AMP responsive element binding protein (CREB), and synaptophysin and hypomethylation of BCL‐2 associated X protein (BAX). These molecular alterations in aging are different from or more subtle than changes associated with AD pathology. The degree to which they are related to changes in cognition or behavior during normal aging remains to be evaluated.     

Magnetic resonance imaging quality and volumes of brain structures from live and postmortem imaging of California sea lions with clinical signs of domoic acid toxicosis

Our goal in this study was to compare magnetic resonance images and volumes of brain structures obtained alive versus postmortem of California sea lions Zalophus californianus exhibiting clinical signs of domoic acid (DA) toxicosis and those exhibiting normal behavior. Proton density-(PD) and T2-weighted images of postmortem-intact brains, up to 48 h after death, provided similar quality to images acquired from live sea lions. Volumes of gray matter (GM) and white matter (WM) of the cerebral hemispheres were similar to volumes calculated from images acquired when the sea lions were alive. However, cerebrospinal fluid (CSF) volumes decreased due to leakage. Hippocampal volumes from postmortem-intact images were useful for diagnosing unilateral and bilateral atrophy, consequences of DA toxicosis. These volumes were similar to the volumes in the live sea lion studies, up to 48 h postmortem. Imaging formalin-fixed brains provided some information on brain structure; however, images of the hippocampus and surrounding structures were of poorer quality compared to the images acquired alive and postmortem-intact. Despite these issues, volumes of cerebral GM and WM, as well as the hippocampus, were similar to volumes calculated from images of live sea lions and sufficient to diagnose hippocampal atrophy. Thus, postmortem MRI scanning (either intact or formalin-fixed) with volumetric analysis can be used to investigate the acute, chronic and possible developmental effects of DA on the brain of California sea lions.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Oxygen restriction of neonate rats elevates neuregulin-1alpha isoform levels: possible relationship to schizophrenia

Neuregulin-1 (NRG-1), a replicated gene in schizophrenia-association studies, exhibits six mRNA-types and two types of the EGF-like domain, alpha and beta. The beta-isoform was extensively studied, less is known about the extent and specific localization of adult brain NRG-1alpha. NRG-1alpha protein levels were reported reduced in postmortem prefrontal-cortex of schizophrenia patients. NRG-1 type I mRNA levels were found higher in postmortem brain in schizophrenia. In an attempt to decipher between a genetic or environmental involvement in the differences in NRG-1 levels in postmortem brain in schizophrenia, and since obstetric complications were suggested non-genetic risk-factors of schizophrenia, we studied the effect of perinatal hypoxia in rats on brain NRG-1alpha protein levels. Seven-day-old rats were exposed to hypoxia versus air. Frontal-cortex levels of NRG-1alpha isoform were quantified at adulthood by Western blotting. Frontal-cortex NRG-1alpha was 32% elevated in hypoxia-exposed rats. The data support the role of non-genetic factors, e.g. oxygen restriction, in the expression of genes associated with schizophrenia.