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β-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aβ peptides, proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (β-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbase(PLUS), employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aβ production.

The diagnosis of dementing disorders is severely hampered by the absence of reliable biomarkers that can be measured in body fluids such as blood, urine and cerebro-spinal fluid (CSF). Searching for biomarkers is hampered by the huge variability between individuals; the use of autopsy specimens induces significant data fluctuation due to rapid post-mortem changes in the specimens. The search for biomarkers obtained from living donors has contributed already a vast amount of data. The role of amyloid and tau as early diagnostic markers in the pathology of dementia has been reported in differential involvement in Alzheimer's disease (AD), late onset Alzheimer disease (LOAD), Lewy Body dementia (DLBD), Vascular dementia, fronto-temporal lobar degeneration (FTLD), Mild cognitive impairment (MCI) and non neurological controls. In the coming decennia, brain/tissue/biobanks (BTB-banks) will have a major role in identifying the relevant biomarkers and will collect, preserve and type RNA and DNA extracted from brain/tissue/body fluids in order to update the pathological hallmarks of dementing disorders. The present paper reviews and compares the currently known/clinically applied biomarkers in dementia which can be identified and incorporated into clinical drug trials and elucidate proposed mechanisms of disease and drug action. Furthermore, the review screens a panel of biomarkers used for early and differential diagnosis and comments on the validity of these biomarkers in reflecting the typical hallmarks of neurological disorders.

The apolipoprotein E (APOE) epsilon4 allele is the best established genetic risk factor for late-onset Alzheimer's disease (LOAD). We conducted genome-wide surveys of 502,627 single-nucleotide polymorphisms (SNPs) to characterize and confirm other LOAD susceptibility genes. In epsilon4 carriers from neuropathologically verified discovery, neuropathologically verified replication, and clinically characterized replication cohorts of 1411 cases and controls, LOAD was associated with six SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. SNP rs2373115 (p = 9 x 10(-11)) was associated with an odds ratio of 4.06 (confidence interval 2.81-14.69), which interacts with APOE epsilon4 to further modify risk. GAB2 was overexpressed in pathologically vulnerable neurons; the Gab2 protein was detected in neurons, tangle-bearing neurons, and dystrophic neuritis; and interference with GAB2 gene expression increased tau phosphorylation. Our findings suggest that GAB2 modifies LOAD risk in APOE epsilon4 carriers and influences Alzheimer's neuropathology.

We used microarrays to compare the gene expression profile in active lesions and donor-matched normal appearing white matter (NAWM) from brain autopsy samples of patients with secondary progressive multiple sclerosis (MS) with that from controls who died from non-neurological diseases. The 123 genes in lesions, and 47 genes in NAWMMS were differentially expressed. Lesions distinguished from NAWMMS by a higher expression of genes related to immunoglobulin synthesis and neuroglial differentiation, while cellular immune response elements were equally dysregulated in both tissue compartments. Current results provide molecular evidence of a continuum of dysfunctional homeostasis and inflammatory changes between lesions and NAWMMS, and support the concept of MS pathogenesis being a generalised process that involves the entire CNS.

The human tuberomamillary nucleus (TMN), that is the sole Source of histamine in the brain, is involved in arousal, learning and memory and is impaired in Alzheimer's disease (AD) as shown by the presence of cytoskeletal alterations, a reduction in the number of large neurons, a diminished neuronal metabolic activity and decreased histamine levels in the hypothalamus and cortex. Experimental data and the presence of sex hormone receptors Suggest an important role of sex steroids in the regulation of the function of TMN neurons. Therefore, we investigated sex-, age- and Alzheimer-related changes in estrogen receptor alpha and beta (ERalpha and ERbeta) in the TMN. In addition, metabolic activity changes of TMN neurons were determined by measuring Golgi apparatus (GA) and cell size. In the present study, ERalpha immunocytochemical expression in AD patients did not differ from that ill elderly controls. However, a larger amount of cytoplasmic ERbeta was found in the TMN cells of AD patients. Earlier studies, using the GA size as a parameter, have shown a clearly decreased metabolic activity in the TMN neurons in AD. In the present study, the size of the GA did not change during aging, indicating the absence of strong metabolic changes. Cell size of the TMN neurons appeared to increase during normal aging in men but not ill women. Concluding, the enhanced cytoplasmic expression of ERbeta in the TMN may be involved in the diminished neuronal metabolism of these neurons in AD patients. (C) 2003 Elsevier B.V. All rights reserved

The neuronal nicotinic receptors (nAChRs) are involved in several processes in brain including nicotine dependence and cognitive disorders. While the number of nAChRs in the brain of tobacco smokers is up-regulated, the receptors are reduced in the brain of patients with Alzheimer's disease (AD). The aim of this study was to investigate nAChR mRNA and protein levels in brain of smoking and non-smoking controls and AD patients. Western blotting and RT-PCR techniques were used to quantify different nAChR subunits in autopsy brain. The alpha4 and alpha7 but not the alpha3 nAChR protein levels were significantly increased in the temporal cortex of smoking (SC) compared with non-smoking controls (NSC). The alpha4-protein level was significantly higher in the temporal cortex of smoking AD (SAD) patients compared with non-smoking AD (NSAD). No changes in the alpha3, alpha4 or alpha7 subunits protein level were found in the hippocampus in any of the smoking groups. For both SADs and NSADs the protein levels for the alpha3 and alpha4 in temporal cortex and hippocampus and alpha7 in the hippocampus were significantly lower compared with non-smoking controls. No significant differences in alpha4 and alpha7 mRNA levels were detected in the hippocampus or temporal cortex of smokers compared with non-smokers. In conclusion this study showed an increased level of alpha4 and alpha7 nAChRs subunits in the temporal cortex of SC compared with NSC. This up-regulation was also seen in SAD although the protein levels of nAChR subunits were still lower in smoking AD brain compared with the NSC. The up-regulation of nAChRs in smoking groups and the loss of these receptors in AD patients were not correlated to any changes at the mRNA level suggesting that these changes may reflect post-transcriptional events.

T-cell accumulation in the central nervous system (CNS) is considered crucial to the pathogenesis of multiple sclerosis (MS). We found that the majority of T cells within the cerebrospinal fluid (CSF) compartment expressed the CXC chemokine receptor 3 (CXCR), independent of CNS inflammation. Quantitative immunohistochemistry revealed continuous accumulation of CXCR3+ T cells during MS lesion formation. The expression of one CXCR3 ligand, interferon (IFN)-gamma-inducible protein of 10 kDa (IP-10)/CXC chemokine ligand (CXCL) 10 was elevated in MS CSF, spatially associated with demyelination in CNS tissue sections and correlated tightly with CXCR3 expression. These data suggest a critical role for CXCL10 and CXCR3 in the accumulation of T cells in the CNS of MS patients.

Post-menopausal estrogen use reduces the risk and severity of Alzheimer's disease (AD). The present study investigates the distribution of both estrogen receptors ER alpha and ER beta in the human hippocampus in aged controls and in AD cases with immunohistochemistry. No ER alpha immunoreactivity was observed both in controls and in AD cases. On the other hand, ER beta was observed in some neuronal cells in the hippocampal subfields CA1--4, in astrocytes and in extracellular deposits both in controls and AD cases. The ER beta immunoreactivity was distinctly increased in all AD cases in cellular and extracellular localizations indicating a role for ER beta-mediated estrogen effects in AD-related neuropathology. This study provides the first demonstration of ER beta in human hippocampus in aged controls compared to AD cases.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a familial neurological disorder, characterized genetically by autosomal dominant inheritance, clinically by behavioral abnormalities and parkinsonism, and neuropathologically by tauopathy. Linkage analyses of affected families have led to identification of several exonic and intronic mutations in the tau gene. In this study, we analyzed molecular species of tau in the soluble and insoluble fractions of brain affected by the FTDP-17 R406W mutation. Protein chemical analysis and Western blotting using site-specific antibodies indicated that almost equal amounts of wild-type and mutant tau were present in the Sarkosyl-insoluble fraction of the R406W brain. Consistent with this, wild-type and mutant tau colocalized in neurofibrillary tangles in the frontal cortex and hippocampus of the R406W brain. In contrast to soluble R406W tau, which was less phosphorylated than soluble wild-type tau, the Sarkosyl-insoluble mutant tau was highly phosphorylated as well as the insoluble wild-type tau.

Zinc has been implicated as a contributing cause of the neuropathology of Alzheimer's disease (AD), but consensus on the zinc content of AD brains has not yet been established. In the present study, multi-element PIXE was used to measure zinc in cryostat sections of brain tissue from AD patients and from normal control subjects. Compared to their age-matched controls, the AD patients showed an increase in zinc in the hippocampal and amygdalar regions. The instrumental PIXE assays do not show whether the zinc changes are due to altered zinc in the boutons of Zinc-ENriched (ZEN) neurons, i.e., zinc ions in synaptic vesicles, or to changes in the amount of zinc tightly bound to macromolecules. We hypothesise that the increased zinc level is caused by an increase in the amount of ZEN terminals. Such an increase could be the result of a sprout of ZEN terminals in diseased areas of the brain.

Oxotremorine-M stimulated [35S]GTP gamma S binding was used to assess acetylcholine muscarinic M2 receptor mediated G-protein function in superior frontal cortical, superior temporal cortical and hippocampal membranes from a series of Alzheimer's disease and matched control subjects. No significant differences were seen in basal [35S]GTP gamma S binding between the groups. The maximal level of oxotremorine-M stimulated [35S]GTP gamma S binding over basal was significantly increased in Alzheimer's disease superior temporal cortex, suggesting an enhanced muscarinic M2 receptor-G-protein coupling efficiency in this region. In contrast, the maximal level of oxotremorine-M stimulated [35S]GTP gamma S binding over basal was unaltered in Alzheimer's disease superior frontal cortex and significantly reduced in Alzheimer's disease hippocampus. Western immunoblotting using antisera towards the alpha-subunits of those G-protein types known to couple muscarinic receptors, revealed that Gq alpha and Gi alpha, but not G(o) alpha, levels were significantly reduced in Alzheimer's disease superior temporal cortex. Neither Gq alpha, Gi alpha, nor G(o) alpha levels were significantly altered in Alzheimer's disease superior frontal cortex or hippocampus. These results suggest that the efficacy of muscarinic M2 receptor G-protein coupling shows regional selective changes in Alzheimer's disease postmortem brain with deficits occurring only in a region that shows severe pathology.

One of the characteristic histological features of Parkinson's disease (PD), with or without dementia, is the presence of Lewy bodies (LBs) in the brainstem and neocortical and limbic structures. They are often accompanied by Alzheimer type pathology (ATP). In the present retrospective study the clinical features and post-mortem findings of 18 consecutive and unselected PD patients were compared, with special reference to the frequent but not exclusive association of LBs with ATP in Lewy body disease (LBD). LBD is the term applied to a particular pattern of neuronal degeneration associated with LBs. In this study of idiopathic PD patients ATP seems to be the major determinant of the cognitive decline in most patients. Cortical Lewy Bodies (CLBs) were present in all patients reviewed, whether or not dementia was present. It was not possible to distinguish a specific pattern in the cognitive or psychopathological symptoms of dementia that would differentiate LBD from Alzheimer's disease (AD). Although in most cases hippocampal CA2-3 ubiquitin immunoreactive neurites were observed, here again there was no correlation with the presence of dementia.

The pharmacological properties of the kappa 1 opioid receptor were investigated in human post-mortem temporal cortical membranes from control and Alzheimer's disease brains, using the kappa 1-selective radioligand [3H]U69593. [3H]U69593 bound to a single high affinity site population with no significant difference between control (Bmax 31 +/- 4.14 fmol/mg protein, KD 1.01 +/- 0.26 nM) and Alzheimer's disease brains (Bmax 37 +/- 4.63 fmol/mg protein, KD 0.86 +/- 0.08 nM). Competition studies with dynorphin B and alpha-neoendorphin gave flat inhibition curves with Hill coefficients of 0.31 +/- 0.04 and 0.49 +/- 0.09 in the control brains and 0.38 +/- 0.05 and 0.48 +/- 0.08 in the Alzheimer's disease brains, respectively. The pI50 values for dynorphin B and alpha-neoendorphin were 8.73 +/- 0.17 and 8.48 +/- 0.09, respectively, in the control brains and 9.30 +/- 0.22 and 8.70 +/- 0.15 in the Alzheimer's disease brains. The guanine nucleotide analogue Gpp(NH)p inhibited binding by ca. 70% in both the control and Alzheimer's disease brains, the residual binding being sensitive to NaCl in both cases. These results indicate that the pharmacological properties and the functional integrity of G-protein coupling of the kappa 1 receptor recognition site are preserved in Alzheimer's disease temporal cortex.

Particulate and soluble guanylyl cyclase activities were studied in postmortem temporal cortex from a series of Alzheimer's disease patients and matched control subjects. Particulate guanylyl cyclase activity was not significantly different between groups. In contrast, the Vmax values for basal and sodium nitroprusside-stimulated soluble guanylyl cyclase activities were approximately 50% lower in the Alzheimer's disease cases, compared to controls. This difference between groups was statistically significant for sodium nitroprusside-stimulated, but not for the basal, enzyme activities. These results provide the first evidence for a loss of nitric oxide responsive guanylyl cyclase activity in Alzheimer's disease brain.

Immunocytochemical staining, using a monoclonal antibody against corticotropin-releasing hormone, was performed on hypothalami of 13 human subjects between 23 and 91 years of age who had not suffered from a primary neurological or psychiatric disease. Corticotropin-releasing hormone (CRH) immunoreactivity was present in neurons of the paraventricular nucleus (PVN) and in their fibers running to the median eminence. The CRH-positive neurons were scattered throughout the PVN, but in the rostral part relatively few cells were present. There were large individual differences in the number and staining intensity of CRH neurons in the PVN and in the staining intensity of the median eminence. These differences seemed not to be attributed to age, sex, postmortem delay, fixation time or hour of death. In the rat, too, no relationship was found between a postmortem delay of up to 24 h and CRH staining intensity of the median eminence. Since the distribution of CRH-immunoreactive neurons in the human PVN strongly overlap with vasopressin, colocalization of these peptides was investigated in a double label study and indeed found in subjects ranging between 43 and 91 years of age. However, cells staining for only one of the peptides were also observed. The vasopressin cells had a mean cellular profile area which was 2.3 times as large as the CRH cells and 2.2 times as large as the CRH and vasopressin containing neurons. In younger subjects (23-37 years of age) no colocalization of the two peptides was seen. The age-dependent colocalization of CRH with vasopressin is interpreted as a sign of increased activation of the CRH neurons with age.

The earlier described age-related decreases in vasopressin innervation of extra-hypothalamic rat brain regions were found to coincide with a decrease in vasopressin expression in the cells where these fibers originated. A significant age-related decrease in the number of vasopressin-immunoreactive cell bodies was found in the medial amygdala and locus coeruleus of senescent Brown-Norway (BN/BiRij) rats (33 months) when compared to middle-aged (19 months) and young (3 months) rats. In addition, total testosterone plasma levels were significantly reduced in middle-aged and old rats as compared to young animals and the number of vasopressin-immunoreactive cells in both the medial amygdala and locus coeruleus correlated significantly with the decreased testosterone levels in a similar way as found earlier for vasopressin terminals.

Immunocytochemical staining of hypothalamic cell groups with four antibodies to Alzheimer paired helical filaments (PHF) (i.e., anti-PHF serum 60e and monoclonal antibody (mAb) Alz-50, both directed against normal and abnormally phosphorylated tau; mAb tau-1, which recognizes tau; and mAb 3-39 to PHF, which recognizes the carboxy terminal domain of ubiquitin) revealed a clear distinction between 12 Alzheimer's disease (AD) patients and seven controls in the hypothalamus. Dystrophic neurites, which appeared to be the most specific components in AD, were most conspicuous after Alz-50 staining. However, Alz-50 also stained neuronal cytoplasm and normal, thin, beaded neurites in the paraventricular nucleus (PVN) of controls, even of young cases. This staining was clearly distinct from the staining of cytoplasm and dystrophic neurites in the PVN of Alzheimer patients. The abundant staining of dystrophic neurites and cell bodies in the nucleus tuberalis lateralis (NTL) in AD, in which no neuronal loss is observed, suggests that alterations in cytoskeletal markers do not necessarily indicate impending cell death. Moreover, the cytoskeletal changes in the NTL, sexually dimorphic and suprachiasmatic nuclei in AD indicate that this condition is not restricted to cortical areas or nuclei projecting to the cortex. Consequently, the pathophysiological implications of cytoskeletal staining in AD are at present far from clear. The human hypothalamus may not only provide a better insight into the pathogenesis of Alzheimer's disease, but could also be of help in the neuropathological diagnosis of this condition.

The coupling of inhibitory guanine nucleotide binding (G i ) proteins to the adenylyl cyclase signal transduction complex was compared in 4 brain regions from a series of Alzheimer's disease and matched control subjects by measuring the inhibition of membrane enzyme activities in response to guanosine 5′-[βγ-imido]diphosphate (Gpp[NH]p) and aluminium fluoride (AlF 4 − ). Basal adenylyl cyclase activities were significantly lower in preparations of angular gyrus and frontal and temporal cortices, but not cerebellum, from the Alzheimer's disease cases compared to controls. Gpp[NH]p and AlF 4 − gave significant inhibitions of adenylyl cyclase activity in all brain regions. The magnitude of these inhibitions, when corrected for altered basal activities, were similar for the Alzheimer's disease and control cases. These results indicate that there is no impairment of G i -protein mediated inhibition of adenylyl cyclase activity in Alzheimer's disease brain.

It has been hypothesized that Alzheimer's disease (AD) is caused by an accumulation of damage in DNA due to defective DNA-repair (21). Attempts to test this hypothesis by determining the activity of DNA-repair systems in nonneuronal cells from AD patients and controls so far provided conflicting results. An alternative approach is the direct comparison of DNA-damage levels in neuronal tissue of AD patients and controls. In the present study we assayed the level of DNA breaks and alkali-labile sites in cerebral cortex tissue samples from AD patients and controls obtained from rapid autopsies. Our data on 11 AD patients and 8 control subjects indicate an at least two-fold higher level of DNA damage in cortex of AD patients as compared to controls.

An immunocytochemical procedure was developed to localize binding sites for vasopressin (VP) in the brain of Brattleboro (di/di) rats after 2 weeks of continuous ventricular administration of the peptide. Accurel-polypropylene tubing loaded with 0.15, 1.5 or 15 micrograms vasopressin was implanted into the lateral ventricle. Subsequently, bound VP was detected immunocytochemically in 2 distinct patterns: in perineuronal structures and dots between cells, in the lateral septum (dorsorostral part), striatum, cingulate cortex, granular cells of the dentate gyrus of the hippocampus, pyramidal cells of CA1 and CA3 hippocampal areas and around cerebellar Purkinje cells. The high dose (15 micrograms) loaded implants revealed the most intense staining; in the cytoplasm of neuronal cell bodies in the lateral and medial septum, striatum, cingulate cortex, bed nucleus of the stria terminalis, organum vasculosum of the laminae terminalis and locus coeruleus. The most intense staining in cell bodies was observed in brains which had low-loaded implants (0.15-1.5 microgram). A variety of controls, proved that no aspecific uptake was involved in the present procedure. The distribution of VP binding sites was only partly coincident with known sites of VP fiber innervation, and largely agrees with data obtained by autoradiographic techniques for [3H]VP binding. The present immunocytochemical technique gave a higher resolution than the currently used autoradiographic techniques. The differences in pattern and intensity of staining due to increasing the dosage rate of the in vivo vasopressin treatment, might mean that the current procedure retains preferentially either low or high affinity populations of binding sites depending on the implanted dose.

Following two weeks of cerebroventricular administration of arginine-vasopressin (AVP) by Accurel implants, two types of binding sites for this peptide were immunocytochemically visualized in blood vessels in the brain of Brattleboro (di/di) rats: (1) endothelial cells of capillaries were stained with the highest density in hippocampus, striatum, and locus coeruleus (LC), whereas only few such stained cells were present in the septum and cerebral cortex. (2) Bound AVP was also present on endothelial cells and pericyte-like cells in larger blood vessels in striatum and the LC. Both types of vasopressin binding site staining on blood vessels were dose-dependent and could be further enhanced by additional in vitro preincubation of the fixed sections with AVP. This staining was not present in rats implanted with either oxytocin or alpha-melanocyte-stimulating hormone.

The metabolism of progesterone in the rat ovary approaching the first ovulation, induced by pregnant mare serum gonadotrophin (PMS)-administration, was examined. In the untreated immature rat, progesterone is metabolized mainly through 5α-reduced pregnanes. 5α-androstane-3α,17β-diol (3α-A) being the main metabolite. Following the first ovulation, 20α-hydroxypregn-4-en-3-one (20α-OHP), and to a lesser extent, 5α-pregnane-3α,20α-diol (allo-P) are the main products. The temporal concentration of 3α-A and of its 3β epimer in peripheral blood of immature rats was measured by RIA. It appears, that in vivo, too. the level of the androstanediols decline before the first ovulation. In order to study this shift in steroid production, the pathways by which the main metabolites are produced were examined several hours before the first ovulation. For this. double labelling and trapping experiments. as well as kinetic studies were performed. The results obtained show that four possible pathways by which 3α-A and allo-P can be formed, co-exist for a short time in the ovary approaching the first ovulation. These results indicate that 5α-reductase. the enzyme responsible for ring A reduction. has a key position in the shift from the “immature” to the “mature” type of steroid metabolism. A considerable decline in 5α-reductase activity was found following administration of PMS: 1 day after the stimulation, a 40% decrease in activity was measured. On the 3rd day. one-tenth of the original activity was detected and by the 7th day only residual activity remained. The collective data obtained in this study explains the shift in steroidogenesis taking place in the ovary at the time of the first ovulation. The importance of these findings to the mechanism of the onset of puberty is discussed.