Ezra Mulugeta

CSF Biomarkers and Incipient Alzheimer Disease in Patients With Mild Cognitive Impairment

CONTEXT Small single-center studies have shown that cerebrospinal fluid (CSF) biomarkers may be useful to identify incipient Alzheimer disease (AD) in patients with mild cognitive impairment (MCI), but large-scale multicenter studies have not been conducted. OBJECTIVE To determine the diagnostic accuracy of CSF beta-amyloid(1-42) (Abeta42), total tau protein (T-tau), and tau phosphorylated at position threonine 181 (P-tau) for predicting incipient AD in patients with MCI. DESIGN, SETTING, AND PARTICIPANTS The study had 2 parts: a cross-sectional study involving patients with AD and controls to identify cut points, followed by a prospective cohort study involving patients with MCI, conducted 1990-2007. A total of 750 individuals with MCI, 529 with AD, and 304 controls were recruited by 12 centers in Europe and the United States. Individuals with MCI were followed up for at least 2 years or until symptoms had progressed to clinical dementia. MAIN OUTCOME MEASURES Sensitivity, specificity, positive and negative likelihood ratios (LRs) of CSF Abeta42, T-tau, and P-tau for identifying incipient AD. RESULTS During follow-up, 271 participants with MCI were diagnosed with AD and 59 with other dementias. The Abeta42 assay in particular had considerable intersite variability. Patients who developed AD had lower median Abeta42 (356; range, 96-1075 ng/L) and higher P-tau (81; range, 15-183 ng/L) and T-tau (582; range, 83-2174 ng/L) levels than MCI patients who did not develop AD during follow-up (579; range, 121-1420 ng/L for Abeta42; 53; range, 15-163 ng/L for P-tau; and 294; range, 31-2483 ng/L for T-tau, P < .001). The area under the receiver operating characteristic curve was 0.78 (95% confidence interval [CI], 0.75-0.82) for Abeta42, 0.76 (95% CI, 0.72-0.80) for P-tau, and 0.79 (95% CI, 0.76-0.83) for T-tau. Cut-offs with sensitivity set to 85% were defined in the AD and control groups and tested in the MCI group, where the combination of Abeta42/P-tau ratio and T-tau identified incipient AD with a sensitivity of 83% (95% CI, 78%-88%), specificity 72% (95% CI, 68%-76%), positive LR, 3.0 (95% CI, 2.5-3.4), and negative LR, 0.24 (95% CI, 0.21-0.28). The positive predictive value was 62% and the negative predictive value was 88%. CONCLUSIONS This multicenter study found that CSF Abeta42, T-tau, and P-tau identify incipient AD with good accuracy, but less accurately than reported from single-center studies. Intersite assay variability highlights a need for standardization of analytical techniques and clinical procedures.

CSF amyloid-beta and tau proteins, and cognitive performance, in early and untreated Parkinson's Disease: the Norwegian ParkWest study

Background Alzheimers disease (AD) pathology is found in a considerable portion of patients with Parkinsons disease (PD), particularly those with early dementia (PDD). Altered cerebrospinal fluid (CSF) levels of amyloid-β (Aβ) and tau proteins have been found in PDD, with intermediate changes for Aβ42 in non-demented PD. The authors investigated whether AD-related CSF protein levels are altered and relate to neuropsychological performance in early, untreated PD. Methods CSF concentrations of Aβ42, Aβ40 and Aβ38 were measured by electrochemiluminiscene and levels of total tau (T-tau) and phosphorylated tau (P-tau) by ELISA in 109 newly diagnosed, unmedicated, non-demented, community-based PD patients who had undergone comprehensive neuropsychological testing, and were compared with those of 36 age-matched normal controls and 20 subjects with mild AD. Results PD patients displayed significant reductions in Aβ42 (19%; p=0.009), Aβ40 (15.5%; p=0.008) and Aβ38 (23%; p=0.004) but not T-tau (p=0.816) or P-tau (p=0.531) compared with controls. CSF Aβ42 reductions in PD were less marked than in AD (53%; p=0.002). Sequential regression analyses demonstrated significant associations between CSF levels of Aβ42 (β=0.205; p=0.019), Aβ40 (β=0.378; p<0.001) and Aβ38 (β=0.288; p=0.001) and memory impairment, but not executive-attentional or visuospatial dysfunction. Tau protein levels did not correlate with cognitive measures. Conclusion CSF Aβ levels are altered in a subset of patients with early PD and relate to memory impairment. Our study suggests that alterations in Aβ protein metabolism may contribute to the heterogeneity in pattern and course of cognitive decline associated with PD. Longitudinal studies are needed to clarify the clinical significance of CSF Aβ peptides as prognostic biomarkers in PD.

Age and diagnostic performance of Alzheimer disease CSF biomarkers

Objectives: Core CSF changes in Alzheimer disease (AD) are decreased amyloid β1–42, increased total tau, and increased phospho-tau, probably indicating amyloid plaque accumulation, axonal degeneration, and tangle pathology, respectively. These biomarkers identify AD already at the predementia stage, but their diagnostic performance might be affected by age-dependent increase of AD-type brain pathology in cognitively unaffected elderly. Methods: We investigated effects of age on the diagnostic performance of CSF biomarkers in a uniquely large multicenter study population, including a cross-sectional cohort of 529 patients with AD dementia (median age 71, range 43–89 years) and 304 controls (67, 44–91 years), and a longitudinal cohort of 750 subjects without dementia with mild cognitive impairment (69, 43–89 years) followed for at least 2 years, or until dementia diagnosis. Results: The specificities for subjects without AD and the areas under the receiver operating characteristics curves decreased with age. However, the positive predictive value for a combination of biomarkers remained stable, while the negative predictive value decreased only slightly in old subjects, as an effect of the high AD prevalence in older ages. Conclusion: Although the diagnostic accuracies for AD decreased with age, the predictive values for a combination of biomarkers remained essentially stable. The findings highlight biomarker variability across ages, but support the use of CSF biomarkers for AD even in older populations.

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors.

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.

Loss of muscarinic M4 receptors in hippocampus of Alzheimer patients.

We assessed muscarinic M(1), M(2) and M(4) receptor subtypes in the hippocampus of Alzheimers and control brains by receptor autoradiography using ligands such as [(125)I]muscarinic toxin-1 ([(125)I]MT-1, M(1) selective), [(3)H]AFDX-384 (M(2) partially selective) and [(125)I]muscarinic toxin 4 ([(125)I]M(4) toxin-1, M(4) selective). Our results revealed a significant decrease in muscarinic M(4) receptor binding in the dentate gyrus and CA4 regions of brain sections from Alzheimers patients compared to controls. No changes in the density of M(1) or M(2) receptor binding were observed. Our findings suggest that, relative to other muscarinic receptor subtypes, the M(4) receptor could be the subtype which is selectively compromised in Alzeheimers disease (AD).

Identification of Novel α-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn1–140) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn1–139 and Ac-α-syn1–103) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).

Inhibition of acetylcholine muscarinic M1 receptor function by the M1‐selective ligand muscarinic toxin 7 (MT‐7)

MT‐7 (1–30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M1 receptor, inhibited the acetylcholine (ACh)‐stimulated [35S]‐guanosine‐5′‐O‐(3‐thio)triphosphate ([35S]‐GTPγS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M1 receptor subtype. MT‐7 failed to affect the ACh‐stimulated [35S]‐GTPγS binding in membranes of CHO cells expressing either the M2, M3 or M4 receptor subtype. In N1E‐115 neuroblastoma cells endogenously expressing the M1 and M4 receptor subtypes, MT‐7 (0.3–3.0 nM) inhibited the carbachol (CCh)‐stimulated inositol phosphates accumulation, but failed to affect the CCh‐induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38‐stimulated cyclic AMP accumulation. In both CHO/M1 and N1E‐115 cells the MT‐7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC50 value. In CHO/M1 cell membranes, MT‐7 (0.05–25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [3H]‐N‐methylscopolamine ([3H]‐NMS) in a concentration‐dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT‐7 (3 nM) decreased the dissociation rate of [3H]‐NMS by about 5 fold. CHO/M1 cell membranes preincubated with MT‐7 (10 nM) and washed by centrifugation and resuspension did not recover control [3H]‐NMS binding for at least 8 h at 30°C. It is concluded that MT‐7 acts as a selective noncompetitive antagonist of the muscarinic M1 receptors by binding stably to an allosteric site.

Neuropsychiatric Correlates of Cerebrospinal Fluid Biomarkers in Alzheimer’s Disease

Background: The aim of this study was to explore the relationship between cerebrospinal fluid biomarkers and neuropsychiatric symptoms in people with Alzheimer’s disease. Psychosis, agitation, apathy and depression were assessed using standardised measures in 32 patients with mild Alzheimer’s disease. Methods: The levels of the 42-amino-acid form of β-amyloid (Aβ1–42), tau and p-tau (phosphorylated at threonine 181) were quantified using the conventional enzyme-linked immunosorbent assay method. Results: Our result shows that apathy is significantly correlated with tau and p-tau but not with Aβ1–42. There were no significant correlations between indices of psychosis/agitation,or depression and cerebrospinal fluid Aβ1–42, tau or p-tau concentrations. Conclusion: Our finding suggests that apathy is associated with the level of neurofibrillary tangles in people with mild Alzheimer’s disease. In contrast, the overall levels of neurofibrillary tangles or amyloid plaques do not seem to be associated with depression or psychosis, indicating that other brain changes contribute to these symptoms.

CSF Amyloid-beta 38 as a novel diagnostic marker for dementia with Lewy bodies.

Background The clinical distinction between Alzheimers disease (AD) and dementia with Lewy bodies (DLB) is sometimes difficult, particularly in mild cases. Although CSF markers such as amyloid β42 (Aβ42) and P-tau can distinguish between AD and normal controls, their ability to distinguish between AD and DLB is not adequate. Objective This study aims to investigate whether CSF markers, in particular levels of Aβ38, can differentiate between mild AD and DLB. Methods 85 individuals were included after standardised diagnostic procedures: 30 diagnosed as probable AD, 23 probable DLB, 20 probable Parkinsons disease dementia and 12 non-demented control subjects. CSF levels of Aβ38, Aβ40 and Aβ42 were determined using commercially available ultra-sensitive multi-array kit assay (MSD) for human Aβ peptides. Total tau (T-tau) and phosphorylated tau (P-tau) were analysed using ELISA (Innotest). In addition, combinations (Aβ42/Aβ38, Aβ42/Aβ40, Aβ42/P-tau and Aβ42/Aβ38/P-tau) were assessed. Results Significant between group differences were found for all CSF measures, and all except Aβ40, Aβ42 and Aβ42/P-tau differed between AD and DLB. The Aβ42/Aβ38 ratio was the measure that best discriminated between AD and DLB (AUC 0.765; p<0.005), with a sensitivity of 78% and a specificity of 67%. Conclusion This study suggests that the level of Aβ38 can potentially contribute in the diagnostic distinction between AD and DLB when combined with Aβ42. Single measures had low diagnostic accuracy, suggesting that developing a panel of markers is the most promising strategy. Studies with independent and larger samples and a priori cut-offs are needed to test this hypothesis.

Estrogen and progesterone treatment: effects on muscarinic M4 receptor subtype in the rat brain

We investigated the effect of ovariectomy (OVX) and hormonal treatment for 10 weeks by estradiol and progesterone on muscarinic M(4) receptor subtype in different brain areas of female rats. Moreover, motor activity of OVX and hormone-treated rats was measured by automated open field exploration boxes. Receptor quantification in the hippocampus, frontal cortex, parietal cortex, amygdala and hypothalamus was done by receptor autoradiography using a selective ligand for muscarinic M(4) receptors. Ovariectomy up-regulated M(4) receptors in the dentate gyrus, CA1, CA3, frontal cortex and hypothalamus whereas the estrogen treatment restored M(4) binding to that of the sham group. Progesterone treatment had no effect on the ovariectomy-induced up-regulation of M(4) receptors. Ovariectomy significantly decreased the exploratory activity of the rats compared to the sham group. Estrogen treatment restored the exploratory behavior of the ovariectomized rats to that of the sham group whereas the progesterone-treated rats were less alert to the surrounding when compared to the sham and estrogen supplemented rats. The effect of estrogen on the hippocampal muscarinic M(4) receptor subtype is a novel finding and may have functional significance for cholinergic receptors especially in relation to postmenopausal memory problems and neurodegenerative disease like Alzheimers disease.