Inga Volkmann

Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer’s disease

A proposed key event in the pathogenesis of Alzheimer’s disease (AD) is the formation of neurotoxic amyloid β (Aβ) oligomers and amyloid plaques in specific brain regions that are affected by the disease. The main plaque component is the 42 amino acid isoform of Αβ (Aβ1-42), which is thought to initiate plaque formation and AD pathogenesis. Numerous isoforms of Aβ, e.g., Aβ1-42, Aβ1-40 and the 3-pyroglutamate derivate of Aβ3-42 (pGluAβ3-42), have been detected in the brains of sporadic AD (SAD) and familial AD (FAD) subjects. However, the relative importance of these isoforms in the pathogenesis of AD is not fully understood. Here, we report a detailed study using immunoprecipitation in combination with mass spectrometric analysis to determine the Aβ isoform pattern in the cerebellum, cortex and hippocampus in AD, including subjects with a mutation in the presenilin (M146V) or amyloid precursor protein (KM670/671NL) genes, SAD subjects and non-demented controls. We show that the dominating Aβ isoforms in the three different brain regions analyzed from control, SAD, and FAD are Aβ1-42, pGluAβ3-42, Aβ4-42 and Aβ1-40 of which Aβ1-42 and Aβ4-42 are the dominant isoforms in the hippocampus and the cortex in all groups analyzed, controls included. No prominent differences in Aβ isoform patterns between FAD and SAD patients were seen, underscoring the similarity in the amyloid pathology of these two disease entities.

Suppression of experimental autoimmune neuritis by ABR-215062 is associated with altered Th1/Th2 balance and inhibited migration of inflammatory cells into the peripheral nerve tissue

The therapeutic effects of ABR-215062, which is a new immunoregulator derived from Linomide, have been evaluated in experimental autoimmune neuritis (EAN), a CD4(+) T cell-mediated animal model of Guillain-Barré syndrome in man. In previous studies, we reported that Linomide suppressed the clinical EAN and myelin antigen-reactive T and B cell responses. Here EAN induced in Lewis rats by inoculation with peripheral nerve myelin P0 protein peptide 180-199 and Freunds complete adjuvant was strongly suppressed by ABR-215062 administered daily subcutaneously from the day of inoculation. ABR-215062 dose-dependently reduced the incidence of EAN, ameliorated clinical signs and inhibited P0 peptide 180-199-specific T cell responses as well as also the decreased inflammation and demyelination in the peripheral nerves. The suppression of clinical EAN was associated with inhibition of the inflammatory cytokines IFN-gamma and TNF-alpha, as well as the enhancement of anti-inflammatory cytokine IL-4 in lymph node cells and periphery nerve tissues, respectively, in a dose-dependent manner. These effects indicate that ABR-215062 may mediate its effects by regulation of Th1/Th2 cytokine balance and suggest that ABR-215062 is potentially a new chemical entity for effective treatment of autoimmune diseases.

Amyloid β-peptide levels in laser capture microdissected cornu ammonis 1 pyramidal neurons of Alzheimerʼs brain

Deposition of the amyloid &bgr;-peptide (A&bgr;) is a pathophysiological event associated with Alzheimers disease. Although much is known about the molecular composition of extracellular A&bgr; deposits, the role of the intracellular pool of A&bgr; is not fully understood. We investigated whether A&bgr; levels are increased in cornu ammonis 1 pyramidal neurons of Alzheimers disease hippocampus, using laser capture microdissection to isolate the neurons and enzyme-linked immunosorbent assay for quantification. Our results showed increased A&bgr;42 levels and an elevated A&bgr;42/A&bgr;40 ratio in neurons from sporadic as well as from familial cases of Alzheimers disease, whereas A&bgr;40 levels remain unchanged between the cases and controls. We speculate that intracellular accumulation of A&bgr;42 increase vulnerability of cornu ammonis 1 pyramidal neurons in Alzheimers disease.

PTEN levels in Alzheimer's disease medial temporal cortex

Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) is a dual (protein tyrosine and lipid) phosphatase one of the functions of which is to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol-3,4-biphosphate thereby inhibiting phosphoinositide-dependent kinase activation of the cell survival kinase Akt. Akt activity is up regulated in Alzheimers disease (AD) brain in parallel to the progression of neurofibrillary pathology. The present study determined whether altered expression of PTEN occurs in Alzheimers disease brain. Western immunoblotting revealed no significant changes of PTEN protein levels in nuclear and membrane fractions of medial temporal cortex from a series of Alzheimers disease and control cases. Similarly, no changes in PTEN protein levels, as determined by dot-blotting, were seen in temporal cortex homogenates from a separate series of Alzheimers disease and control brains. A small but significant decrease in the levels of Ser(380) p-PTEN was seen in homogenates of Alzheimers disease temporal cortex. Immunohistochemistry revealed PTEN immunoreactivity in a number of brain structures including neurons, capillaries and structures resembling oligodendrocytes and astrocytes. The majority of temporal cortex pyramidal neurons (93-100%) were PTEN immunopositive. The Alzheimers disease cases had significantly lower numbers of total ( approximately 12% loss, P<0.02) and PTEN immunopositive ( approximately 15% loss, P<0.01) pyramidal neurons as compared to the control cases.

Growth-associated protein GAP-43 in the frontal cortex and in the hippocampus in Alzheimer's disease: an immunohistochemical and quantitative study.

Summary. We studied the growth-associated protein, GAP-43 (also called neuromodulin and B-50) in post-mortem brain tissue using immunohistochemistry and quantitative Western blotting, from patients with Alzheimers disease (AD) and age-matched control subjects. By immunohistochemistry, we found a clear reduction of GAP-43 in the frontal cortex, while in the hippocampus, there was a marked reduction in some areas (dentate molecular layer, stratum moleculare and radiale of CA1 and CA4), while not in other areas (stratum lacunosum, pyramidale and oriens of CA1). Moreover, in the hippocampus, neuritic staining was prominent, and was often associated with senile plaques. Quantitative analysis showed that GAP-43 was significantly reduced in AD, both in the frontal cortex (70% of the control value, p < 0.01) and in the hippocampus (81% of the control value, p < 0.05). In the frontal cortex, there was a significant negative correlation between GAP-43 and duration of dementia (r = −0.58; p < 0.02) and a positive correlation between GAP-43 and the synaptic vesicle-specific protein rab3a (r = 0.62; p < 0.05), while no such correlation were found in the hippocampus. In contrast, a significant positive correlation was found between GAP-43 and the number of senile plaques in the hippocampus (r = 0.64; p < 0.05), but not in the frontal cortex. GAP-43 is known to be involved in maintenance of synapses and in neuritic regeneration. Our findings may suggest that in the frontal cortex, GAP-43 levels decline as a consequence of the synaptic degeneration, while in the hippocampus, sprouting processes, involving GAP-43, are active.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade enhances incidence and severity of experimental autoimmune neuritis in resistant mice

Experimental autoimmune neuritis (EAN), an autoimmune inflammatory demyelinating disease of the peripheral nervous system, represents an animal model of the human Guillain-Barré syndrome. EAN can be induced by active immunization in several animals, including Lewis rats. In contrast, most strains of mice including the widely used C57BL/6 (B6) strain are reputedly resistant to the induction of EAN. In the present study, we demonstrate that in B6 mice, anti-CTLA-4 monoclonal antibody administration in conjunction with immunization with the P0 protein derived peptide 180-199 can induce clinical and pathological definite EAN. Upregulating effects of CTLA-4 blockade on initial and ongoing EAN are demonstrated. CTLA-4 blockade augmented cellular infiltration and enhanced demyelination in the target organ sciatic nerves as well as increased T cell proliferation in lymph node cells. Moreover, serum levels of IFN-gamma and IL-4 were increased. Thus, manipulation of CTLA-4/B7 costimulatory pathway by CTLA-4 blockade can promote autoreactivity and break the relative tolerance to peripheral autoantigen P0 in resistant B6 mice.

CD147, a γ-secretase associated protein is upregulated in Alzheimer's disease brain and its cellular trafficking is affected by presenilin-2

Gamma-secretase activity has been extensively investigated due to its role in Alzheimers disease. Here, we studied the association of CD147, a transmembrane glycoprotein belonging to the immunoglobulin family, with gamma-secretase and its expression in Alzheimers disease and control tissues. Subcellular fractionation of postmitochondrial supernatant from rat brain on step iodixanol gradient in combination with co-immunoprecipitation using an anti-nicastrin antibody showed association of limited amount of CD147 to gamma-secretase. By immunoblotting of postnuclear pellets from Alzheimers disease and control human brain tissues we showed that CD147 with molecular weight 75 kDa is upregulated in frontal cortex and thalamus of the Alzheimers disease brains. Immunohistochemistry of brain tissues from Alzheimers disease and control revealed specific upregulation of CD147 in neurons, axons and capillaries of Alzheimers disease frontal cortex and thalamus. The effect of presenilin-1 and -2, which are the catalytic subunits of gamma-secretase, on CD147 expression and subcellular localization was analyzed by confocal microscopy in combination with flow cytometry and showed that PS2 affected the subcellular localization of CD147 in mouse embryonic fibroblast cells. We suggest that a small fraction of CD147 present in the brain is associated with the gamma-secretase, and can be involved in mechanisms dysregulated in Alzheimers disease brain.

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study.

β‐amyloid (Aβ) is the main constituent of senile plaques seen in Alzheimers disease. Aβ is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases β‐ and β‐secretase. In this study, we examined content and localization of β‐secretase‐cleaved APP (β‐sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular β‐sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. β‐sAPP was found to be localized in astrocytes and in axons. We found the β‐sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal β‐sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. β‐sAPP was also found surrounding senile plaques and cerebral blood vessels. The results presented here show altered β‐sAPP staining in the AD brain, suggestive of abnormal processing and transport of APP.

Analysis of microdissected neurons by 18O mass spectrometry reveals altered protein expression in Alzheimer's disease.

It is evident that the symptoms of Alzheimers disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post‐mortem brains by laser capture microdissection. By using 18O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat‐shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron‐specific analysis. Hence, we conclude that cell‐specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis.

Characterization and quantification of 125I-bolton hunter substance P binding sites in human brain

(125)I-Bolton Hunter substance P (BHSP) specific binding sites in human brain synaptosomal membrane preparations were studied with respect to binding characteristics and regional distribution. Specific binding of (125)I-BHSP, which constituted 75% of total binding, and 7% of the total radioactivity added to the incubation medium, was saturable and stable at 20 degrees C. Scatchard analysis of data from equilibrium studies revealed the existence of a single class of binding sites (K(D) = 0.37 nM, B(MAX) = 47.3 fmol/mg tissue protein) in human nucleus caudatus. Competitive studies of (125)I-BHSP binding in human nucleus caudatus gave the following rank order of potencies among tachykinins: substance P > physalaemin > neurokinin A ? neurokinin B ? kassinin ? eledoisin. Measurements of (125)I-BHSP receptor binding densities in different regions of autopsy human brain revealed highest densities in the corpus of nucleus caudatus and the putamen. Moderately high binding densities were detected in the amygdala, hypothalamus, caput of nucleus caudatus and certain parts of the frontal lobe, whereas other regions showed only low levels of binding.