Mike Beck

Phylogenetic diversity of the expression of the microtubule-associated protein tau: implications for neurodegenerative disorders.

The microtubule-associated protein tau regulates the dynamic stability of the neuronal cytoskeleton by interacting with microtubules. It is encoded by a single gene, but expressed in a variety of isoforms due to differential RNA splicing. Six isoforms can be found in the human central nervous system. These isoforms differ in their ability to promote the assembly of microtubules as well as in their capacity to stabilize existing microtubule structures. Furthermore, some of the isoforms of tau are specifically involved in the pathogenesis of neurodegenerative disorders. Thus, splicing of tau might critically influence the physiological functions of tau protein as well as the pathogenesis of neurodegenerative diseases with tauopathy. The present study addresses the differential expression of the six isoforms of tau in the central nervous system of 12 mammalian species including Homo sapiens. The occurrence of each of the six tau isoforms was highly variable. However, species that were phylogenetically related expressed a similar pattern of tau isoforms. These results suggest a phylogenetic descent of splicing paradigms, which can be matched with known phylogenetic concepts based on morphological and molecular genetical studies. Especially, the unique expression pattern of tau isoforms in the human central nervous system implicates a possible link to the particular vulnerability of humans to neurodegenerative disorders with tauopathy, namely Alzheimers disease, frontotemporal dementia and Picks disease.

Constitutive overactivation of protein kinase C in guinea pig brain increases α-secretory APP processing without decreasing β-amyloid generation

Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and β‐amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA‐methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester‐stimulated PKC enzyme activity (by 47% and 71%) as compared to age‐matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol‐treated animals, there was increased α‐secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP α‐secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of β‐amyloid peptides 1–40 or 1–42 as measured by ELISA, suggesting that only the α‐secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.

EXPRESSION OF AMYLOID PRECURSOR PROTEIN MRNA ISOFORMS IN RAT BRAIN IS DIFFERENTIALLY REGULATED DURING POSTNATAL MATURATION AND BY CHOLINERGIC ACTIVITY

Pathological processing of the amyloid precursor protein (APP) is assumed to be responsible for the amyloid deposits in Alzheimer‐diseased brain tissue, but the physiological function of this protein in the brain is still unclear. The aim of this study is to reveal whether the expression of different splicing variants of APP transcripts in distinct brain regions is driven by postnatal maturation and/or regulated by cortical cholinergic transmission, applying quantitative in situ hybridization histochemistry using 35S‐labeled oligonucleotides as specific probes to differentiate between APP isoforms. In cortical brain regions, the expression of both APP695 and APP751 is high at birth and exhibits nearly adult levels. The developmental expression pattern of cortical APP695 displays a peak value around postnatal day 10, while the age‐related expression of APP751 demonstrates peak values on postnatal days 10 and 25, with the highest steady state levels of APP751 mRNA on day 25. During early development, the cortical laminar distribution of the APP695, but not APP751, mRNA transiently changes from a more homogeneous distribution at birth to a pronounced laminar pattern with higher mRNA levels in cortical layer III/IV detectable at the age of 4 days and persisting until postnatal day 10. The distinct age‐related changes in cortical APP695 and APP751 mRNA levels reflect the functional alterations during early brain maturation and suggest that APP695 might play a role in establishing the mature connectional pattern between neurons, whereas APP751 could play a role in controlling cellular growth and synaptogenesis.Lesion of basal forebrain cholinergic system by the selective cholinergic immunotoxin 192IgG‐saporin resulted in decreased levels of APP695 but not APP751 and APP770 transcripts by about 15–20% in some cortical (cingulate, frontal, parietal, piriform cortex), hippocampal regions (CA1, dentate gyrus), and basal forebrain nuclei (medial septum, vertical limb of diagonal band), detectable not earlier than 30 days after lesion and persisting until 90 days postlesion, suggesting that the nearly complete loss of cortical cholinergic input does not have any significant impact on the expression of APP mRNA isoforms in cholinoceptive cortical target regions.

Guinea Pigs as a Nontransgenic Model for APP Processing in Vitro and in Vivo

Alzheimers disease (AD) is characterized, amongst others, by the appearance of vascular and parenchymal β-amyloid deposits in brain. Such aggregates are mainly composed of β-amyloid peptides, which are derived by proteolytic processing of a larger amyloid precursor protein (APP). APP is highly conserved among mammalian species, but experimental studies in rodents are often hampered by the humble APP-processing in the amyloidogenic pathway and by the inability of rodent β-amyloid peptides to form higher molecular aggregates such as soluble oligomers and insoluble β-amyloid plaques. Thus, there is need for in vitro and in vivo model systems that allow identification of factors that increase amyloidogenic APP processing and accelerate β-amyloid plaque formation and testing the potency of pharmacological manipulations to ameliorate β-amyloid load in brain. Transgenic mice that overexpress human APP containing AD-associated mutations that favor the amyloidogenic pathway of APP processing represent such a model. However, mutations of the APP gene are not frequent in AD and, therefore, the mechanisms of β-amyloid plaque formation, the composition of β-amyloid plaques, and the accompanying tissue response in brain of these animals may be different from that in AD. In contrast, guinea pigs express β-amyloid peptides of the human sequence and appear to represent a more physiological model to examine the long-term effects of experimental manipulations on APP processing and β-amyloid plaque formation in vivo. Additionally, APP processing in guinea pig primary neuronal cultures has been shown to be similar to cultures of human origin. In this article we highlight the advantages and limitations of using guinea pigs as experimental models to study APP processing.

Amyloid precursor protein in guinea pigs — complete cDNA sequence and alternative splicing

We present the cDNA sequence of the guinea pig amyloid precursor protein comprising the complete coding sequence of 770 amino-acid residues. By alternative splicing of three exons transcripts encoding for 695, 714 and 751 amino acids and all forms previously denoted as L-APP are also generated. Guinea pig amyloid precursor protein was shown to exhibit extensive sequence similarity to its human and murine homologues of approx. 97% at the protein level which implies an evolutionary conserved but yet not fully understood physiological function.

Modulation of APP processing and secretion by okadaic acid in primary guinea pig neurons

Summary. Primary cultures of guinea pig neurons were used as a model system to study the influence of the protein phosphatase inhibitor okadaic acid (OA) on the secretion, processing and phosphorylation of the amyloid precursor protein (APP). This primary cell culture system mimics more closely than other cell culture systems the human in vivo condition, as guinea pig APP is 98% homologous to human APP at the protein level, identical regarding the Aβ sequence and is processed in a similar manner as human APP. Both intracellular and secreted APP was upregulated by OA treatment (0.3 nM–10 nM) of 14 days old cultures in a concentration dependent manner while the amount of Aβ in the medium was decreased. OA treatment did not affect cell membrane integrity of primary neurons but induced DNA fragmentation. Phosphorylation of APP was unchanged by the low OA concentration used. These results show that OA treatment of guinea pig primary cultures might be used as a model to study the effects of modulation of signal transduction on secretion and processing of APP.

MK 801 attenuates c-Fos and c-Jun expression after in vitro ischemia in rat neuronal cell cultures but not in PC 12 cells

Abstract Cellular homeostatic adaptation to cerebral ischemia is complex and contains changes in receptor mediated gene expression and signaling pathways. The proteins of the immediate early genes c-Fos and c-Jun are thought to be involved in coupling neuronal excitation to target gene expression, due to formation of heterodimers and binding to the AP1 promotor region. We used an in vitro model to compare ischemia induced c-Fos and c-Jun expression in rat neuronal cell cultures and nerve growth factor (NGF) differentiated PC 12 cells. Since activation of glutamate receptors is known to mediate ischemic injury we determined the effect of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK 801 on c-Fos and c-Jun expression in both cell culture systems during ischemia. Neuron rich cultures and NGF differentiated PC 12 cells were exposed to sublethal in vitro ischemia using an hypoxic chamber flushed with argon/CO (95%/5%). C-Fos and c-Jun mRNA expression was analyzed by competitive reverse 2 transcription-polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standard. One hour of in vitro ischemia significantly increased c-Fos and c-Jun mRNA levels in both cell culture systems. In neuron rich cultures a 10-fold (c-Fos) and 7-fold (c-Jun) mRNA increase was observed. The mRNA rise was less pronounced in PC 12 cells (5.5-fold and 2-fold) for c-Fos and c-Jun, respectively. The addition of MK 801 significantly reduced the expression of c-Fos and c-Jun mRNA in neuronal cultures, whereas no effect was detectable in PC 12 cells. Since MK 801 failed to reduce the c-Fos and c-Jun expression in NGF differentiated PC 12 cells different signaling pathways may initiate c-Fos and c-Jun expression in both cell culture systems.

Guinea-pig primary cell cultures provide a model to study expression and amyloidogenic processing of endogenous amyloid precursor protein.

Until now guinea-pigs have been rarely used to investigate formation and deposition of Alzheimers disease-associated amyloid beta peptides despite the sequence identity of human and guinea-pig amyloid beta peptides being known, and the overall similarity of human and guinea-pig amyloid precursor protein. We now describe a primary cell culture system of mixed fetal guinea-pig brain cells, which we have applied to characterize endogenous amyloid precursor protein processing and amyloid beta formation. These cell cultures were established at embryonic day 24 of guinea-pigs after comparison of selected stages of guinea-pig ontogenetic development with the known ontogeny of rats, and were characterized by immunocytochemical detection of neuronal and glial marker proteins. Amyloid precursor protein expression, processing and amyloid beta formation increased in parallel with cellular maturation during cultivation and reached a stable phase after approximately 14 days in vitro therefore providing a suitable time for analysis. Aged cultures display strong neuronal amyloid precursor protein immunoreactivity and an altered profile of amyloid precursor protein isoform messenger RNA expression due to glial proliferation as single neurons were shown to retain their typical pattern of amyloid precursor protein expression. We show that amyloid precursor protein in guinea-pig cells is processed by different protease activities which most likely represent alpha- and beta-secretase, leading to the generation of soluble amyloid precursor protein derivatives. Furthermore, endogenous amyloid precursor protein processing leads to production of substantial amounts of amyloid beta-peptides which accumulate in conditioned culture medium. Amyloid beta was readily detectable by western blot analysis and was shown to consist of approximately 80-90% amyloid beta(1-40). We suggest that primary guinea-pig cell cultures provide a valuable tool in amyloid research that resembles amyloid precursor protein processing under physiological concentrations and, therefore, the situation in humans more closely than current rodent models. It should be especially useful in screening experiments for secretase inhibiting compounds.

Altered phosphofructokinase mRNA levels but unchanged isoenzyme pattern in brains from patients with Alzheimer's disease

In order to find out whether the increased phosphofructokinase (PFK) activities observed in brains from Alzheimers disease (AD) patients are associated with alterations in PFK mRNA levels, we determined total PFK mRNA and the three different PFK isoenzyme mRNAs in AD and control patients by ribonuclease protection assay (RPA) and quantitative RT-PCR. PFK mRNA levels were found increased in some brain areas in AD patients. While all three PFK isoenzyme mRNAs were detectable in every studied brain sample, no changes of the PFK isoenzyme pattern were observed in patients with AD.

Analysis of the molecular heterogeneity of the microtubule-associated protein tau by two-dimensional electrophoresis and RT-PCR.

The microtubule-associated protein tau is a member of a group of proteins, promoting assembly and stabilization of microtubules. In several tauopathic neurodegenerative disorders, namely Alzheimers and Picks disease and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP 17) this protein is converted into fibrilar polymers which form the component of insoluble proteanous deposits such as neurofibrillary tangles. The formation of these fibrils is believed to interrupt the physiological function of neurons resulting in degeneration and cell death. Tau protein exists as a family of heterogeneous isoforms derived by both, differential splicing of tau-mRNA and posttranslational modification of the protein. Since the role of the different isoforms during the process of neurodegeneration is not well understood and as their balance might be altered in some cases of tauopathies (Spillantini et al., Proc. Natl. Acad. Sci. USA 1998;95:7737-7741), the detailed analysis of the molecular heterogeneity gained outstanding interest. The method presented here allows the analysis of both, differential splicing and phosphorylation of tau protein by the application of two-dimensional (2D) electrophoresis and Western blot detection. Tau protein isoforms could be identified from the 2D pattern of dephosphorylated tau in concordance with the results of tau-mRNA analysis by RT-PCR. The protocol presented was successfully applied to analysis of tau isoforms of human brain (Janke et al., FEBS Lett. 1996;379:222-226) and of several species, revealing a phylogenetic correlation of tau protein patterns in mammals (Janke et al., Mol. Brain Res. 1999;68:119-128). The present paper provides a detailed description of the technique and discusses its prospects and limits.