Ralf Peter Meyer

Murine features of neurogenesis in the human hippocampus across the lifespan from 0 to 100 years.

Background Essentially all knowledge about adult hippocampal neurogenesis in humans still comes from one seminal study by Eriksson et al. in 1998, although several others have provided suggestive findings. But only little information has been available in how far the situation in animal models would reflect the conditions in the adult and aging human brain. We therefore here mapped numerous features associated with adult neurogenesis in rodents in samples from human hippocampus across the entire lifespan. Such data would not offer proof of adult neurogenesis in humans, because it is based on the assumption that humans and rodents share marker expression patterns in adult neurogenesis. Nevertheless, together the data provide valuable information at least about the presence of markers, for which a link to adult neurogenesis might more reasonably be assumed than for others, in the adult human brain and their change with increasing age. Methods and Findings In rodents, doublecortin (DCX) is transiently expressed during adult neurogenesis and within the neurogenic niche of the dentate gyrus can serve as a valuable marker. We validated DCX as marker of granule cell development in fetal human tissue and used DCX expression as seed to examine the dentate gyrus for additional neurogenesis-associated features across the lifespan. We studied 54 individuals and detected DCX expression between birth and 100 years of age. Caveats for post-mortem analyses of human tissues apply but all samples were free of signs of ischemia and activated caspase-3. Fourteen markers related to adult hippocampal neurogenesis in rodents were assessed in DCX-positive cells. Total numbers of DCX expressing cells declined exponentially with increasing age, and co-expression of DCX with the other markers decreased. This argued against a non-specific re-appearance of immature markers in specimen from old brains. Early postnatally all 14 markers were co-expressed in DCX-positive cells. Until 30 to 40 years of age, for example, an overlap of DCX with Ki67, Mcm2, Sox2, Nestin, Prox1, PSA-NCAM, Calretinin, NeuN, and others was detected, and some key markers (Nestin, Sox2, Prox1) remained co-expressed into oldest age. Conclusions Our data suggest that in the adult human hippocampus neurogenesis-associated features that have been identified in rodents show patterns, as well as qualitative and quantitative age-related changes, that are similar to the course of adult hippocampal neurogenesis in rodents. Consequently, although further validation as well as the application of independent methodology (e.g. electron microscopy and cell culture work) is desirable, our data will help to devise the framework for specific research on cellular plasticity in the aging human hippocampus.

Localization and characterization of cytochrome P450 in the brain. In vivo and in vitro investigations on phenytoin- and phenobarbital-inducible isoforms.

The antiepileptic drug phenytoin is known to be substrate as well as inducer of cytochrome P450 (P450) in the mammalian liver. We were able to show the expression of P450 species immunorelated to the main phenytoin-induced hepatic isoforms in mice (CYP2C29) and rats (CYP2B1,2) also in the central and peripheral nervous system and primary cultures of cell types from the brain. The 2B1,2 related protein showed only a weak constitutive expression in vivo and in vitro analyzed by immunocytochemistry, in situ hybridization, Northern blot and RT/polymerase chain reaction (PCR). Contrary, the CYP2C29 related form is inducible by phenytoin at about 1.5-fold starting from an already higher constitutive level. This protein is characterized by a remarkable tendency to dissociate from the endomembranes during tissue homogenization. The supernatant of microsomal pellet is able to metabolize phenytoin in a reconstitutive system.

Possible function of astrocyte cytochrome P450 in control of xenobiotic phenytoin in the brain: in vitro studies on murine astrocyte primary cultures.

[4-(14)C]Phenytoin underwent a rapid cellular uptake by diffusion within 5 min when applied in a concentration of 10 microM to mouse brain astrocyte cultures. Subsequently, a slow linear increase of intracellular radioactivity indicated metabolic trapping of the drug, with final concentrations reaching 144 pmol phenytoin/mg protein in the astrocytes. Phenytoin levels from 1 to 10 microM decreased cell viability by 15%. The action of cytochrome P450 present in astrocytes in concentrations of 16-17 pmol P450/mg protein could explain these slight cytotoxic effects by generating intermediate metabolites of phenytoin. In contrast, concentrations of 50 microM strongly inhibited cell proliferation. A Cyp2c29 immunorelated P450 isoform was expressed in nearly all astrocytes in culture. Intracellular [4-(14)C]phenytoin was degraded to its major metabolites dihydrodiol, p-HPPH, and m-HPPH through a P450-dependent reaction with a specific activity of 0.66 pmol/min x mg protein, or 0.12 pmol/min x mg protein as measured in cell homogenates. These data underscore the importance of astrocytes as brain cells active in the detoxification of foreign substrates, but also in their toxification due to reactive metabolites generated during these metabolic processes. After diffusionary influx of drugs and other xenobiotics, the astrocyte P450 monooxygenases perform an essential role in the mediation of toxicity most frequently encountered in highly vulnerable neurons.

Anti-epileptic drug phenytoin enhances androgen metabolism and androgen receptor expression in murine hippocampus

Epilepsy is very often related to strong impairment of neuronal networks, particularly in the hippocampus. Previous studies of brain tissue have demonstrated that long‐term administration of the anti‐epileptic drug (AED) phenytoin leads to enhanced metabolism of testosterone mediated by cytochrome P450 (CYP) isoforms. Thus, we speculate that AEDs affect androgen signalling in the hippocampus. In the present study, we investigated how the AED phenytoin influences the levels of testosterone, 17β‐oestradiol, and androgen receptor (AR) in the hippocampus of male C57Bl/6J mice. Phenytoin administration led to a 61.24% decreased hippocampal testosterone level as compared with controls, while serum levels were slightly enhanced. 17β‐Oestradiol serum level was elevated 2.6‐fold. Concomitantly, the testosterone metabolizing CYP isoforms CYP3A11 and CYP19 (aromatase) have been found to be induced 2.4‐ and 4.2‐fold, respectively. CYP3A‐mediated depletion of testosterone‐forming 2β‐, and 6β‐hydroxytestosterone was significantly enhanced. Additionally, AR expression was increased 2‐fold (mRNA) and 1.8‐fold (protein), predominantly in the CA1 region. AR was shown to concentrate in nuclei of CA1 pyramidal neurons. We conclude that phenytoin affects testosterone metabolism via induction of CYP isoforms. The increased metabolism of testosterone leading to augmented androgen metabolite formation most likely led to enhanced expression of CYP19 and AR in hippocampus. Phenytoin obviously modulates the androgen signalling in the hippocampus.

Modulation of androgen and estrogen receptor expression by antiepileptic drugs and steroids in hippocampus of patients with temporal lobe epilepsy

Purpose:  Many of the antiepileptic drugs (AED) used in therapy of temporal lobe epilepsy (TLE) are known as cytochrome P450 (CYP, P450) inducers. These AEDs are thought to modulate androgen and estrogen pathways in hippocampus, and therefore cause mental and reproductive disorders found in TLE patients. In the present study, we analyzed expression of androgen receptor (AR), estrogen receptor α (ERα), and CYP3A in the hippocampus of TLE patients and in murine hippocampal cell line HN25.1.

7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver

The cytochrome P450 subfamily CYP3A belongs to the most important detoxification enzymes. Because the main CYP3A isoforms are not polymorphic and therefore detract themselves from genetic screening as a potent prediction marker for drug metabolism or induction effects, effective in vitro testing of a putative drug-CYP3A interaction is indicated. We used mouse liver microsomes treated with the model drug phenytoin to set up an effective and reliable in vitro test system. A metabolic assay analyzing 7-alkoxyresorufin-O-dealkylation showed specific CYP3A-dependent 7-benzyloxyresorufin oxidation (BROD). This was confirmed by testing other alkoxyresorufins (7-ethoxy-, 7-methoxy-, and 7-pentoxyresorufin) in mice and correlation of the data with testosterone 6beta-hydroxylation and a plethora of isoform-specific chemical inhibitors (orphenadrine, chloramphenicol, nifedipine, ketoconazole, and sulfaphenazole). Isoform-specific expression and induction of CYP3A11 in mouse liver was tested by RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR), and immunoblot. With the BROD assay, we could clearly dissect CYP3A11 from other P450s induced by phenytoin-like CYP2C29, CYP2B9, CYP1A1, and CYP4A. We conclude that the BROD assay is a specific tool to assign CYP3A induction by drugs or other chemicals, at least in a mouse model system.

Concordant up-regulation of cytochrome P450 Cyp3a11, testosterone oxidation and androgen receptor expression in mouse brain after xenobiotic treatment

Inactivation of testosterone by specific hydroxylations is a main function of cytochrome P450 (P450, CYP) in the brain. Recent data imply that induction of brain P450s by neuroactive drugs alters steroid hormone levels and endocrine signalling, giving rise to endocrine disorders. In this study, we investigated this drug–hormone crosstalk in mouse brain. Phenytoin led to a significant increase of 2α‐, 2β‐, 6β‐, 16α‐ and 16β‐hydroxytestosterones, while 6α‐ and 15α‐hydroxytestosterones showed no significant alteration of their metabolism compared with untreated controls. Inhibition of testosterone hydroxylation using the chemical inhibitors orphenadrine, chloramphenicol, ketoconazole and nifedipine as well as antibodies against CYP3A‐ and 2B‐isoforms pointed to major role of Cyp3a11 and an only minor function of Cyp2b9/10 in mouse brain. Cyp3a11 revealed to be the major isoform affected by phenytoin. There was considerable overlap of Cyp3a11 and AR expression in neuronal structures of the limbic system, namely the hippocampus, amygdala, hypothalamus and thalamus. Phenytoin treatment led to an increase of both, Cyp3a11 and AR expression in the limbic system. Additionally, the coherence between CYP3A and AR expression was analysed in PC‐12 cells. Inhibition of phenytoin‐induced endogenous CYP3A2 and AR by ketoconazole led a reduction of their expression to basal levels. We conclude that Cyp3a11 plays a crucial role in directing drug action to hormonal response within the limbic system of mouse brain in a so‐called drug–hormone crosstalk.

Antiepileptic Drugs Affect Neuronal Androgen Signaling via a Cytochrome P450-Dependent Pathway

Recent data imply an important role for brain cytochrome P450 (P450) in endocrine signaling. In epileptic patients, treatment with P450 inducers led to reproductive disorders; in mouse hippocampus, phenytoin treatment caused concomitant up-regulation of CYP3A11 and androgen receptor (AR). In the present study, we established specific in vitro models to examine whether CYP3A isoforms cause enhanced AR expression and activation. Murine Hepa1c1c7 cells and neuronal-type rat PC-12 cells were used to investigate P450 regulation and its effects on AR after phenytoin and phenobarbital administration. In both cell lines, treatment with antiepileptic drugs (AEDs) led to concomitant up-regulation of CYP3A (CYP3A11 in Hepa1c1c7 and CYP3A2 in PC-12) and AR mRNA and protein. Inhibition of CYP3A expression and activity by the CYP3A inhibitor ketoconazole or by CYP3A11-specific short interfering RNA molecules reduced AR expression to basal levels. The initial up-regulation of AR signal transduction, measured by an androgen-responsive element chloramphenicol-acetyltransferase reporter gene assay, was completely reversed after specific inhibition of CYP3A11. Withdrawal of the CYP3A11 substrate testosterone prevented AR activation, whereas AR mRNA expression remained up-regulated. In addition, recombinant CYP3A11 was expressed heterologously in PC-12 cells, thereby eliminating any direct drug influence on the AR. Again, the initial up-regulation of AR mRNA and activity was reduced to basal levels after silencing of CYP3A11. In conclusion, we show here that CYP3A2 and CYP3A11 are crucial mediators of AR expression and signaling after AED application. These findings point to an important and novel function of P450 in regulation of steroid hormones and their receptors in endocrine tissues such as liver and brain.

Effect of phenytoin on cytochrome P450 2B mRNA expression in primary rat astrocyte cultures

Studies on cytochrome P450 2B (CYP2B) in the brain have essentially been focused on protein characterization and regional distribution. Due to the high sequence homology between the closely related CYP2B1 and 2B2 isoforms and the low amounts of the corresponding mRNAs few efforts have been made to analyze the expression, regulation, and inducibility of these P450 genes in a specific cell type. In the present study, we investigated CYP2B mRNA expression in primary rat astrocyte cultures under the influence of the anti‐epileptic drug phenytoin, which is known to be a CYP2B inducing agent in liver. In situ hybridization with a digoxigenin (DIG)‐labeled cRNA probe demonstrated that 30–40% of the astrocytes strongly expressed a CYP2B mRNA‐specific signal within the first week of cultivation. With increasing age (>14 days) a greater percentage of cells (>90%) expressed mRNA for P450 2B. However, the level of transcriptional activity was substantially lower than in younger cultures. To discriminate between the 2B1 and 2B2 isoforms the reverse transcription/ polymerase chain reaction (RT/ PCR) procedures were proved for rat hepatic mRNA as a control assay. Subsequently, the application of this method on cultured astrocytes confirmed that these brain cells may express CYP2B1 mRNA. CYP2B2 mRNA could not be detected in astrocyte cultures at any age examined. Phenytoin led to the down regulation of CYP2B1 mRNA, which contrasts with the drug inducing effect on hepatic CYP2B1 and 2B2 levels. After 4 hr of exposure of phenytoin to the astrocytes no amplification product could be detected at all. Phenytoin did not induce either CYP2B1 or 2B2 expression. J. Neurosci. Res. 54:402–411, 1998.

A role for CYP in the drug–hormone crosstalk of the brain

Importance of the field: Libido disorders, menstruation problems, perception and cognition deficiencies are potentially undesired, but clinically emerging, side effects after long-term therapy with antiepileptic drugs or chemotherapeutic agents. These disorders were shown to occur predominantly in patients who had taken drugs interacting with the CYP system of the brain, particularly in the hippocampus, the hypothalamus, the cerebellum or in the amygdala. CYPs present and active in these regions usually inactivate steroid hormones such as testosterone. Areas covered in this review: The present review focuses on recent concepts of brain CYP function, gives an outlook on neuroactive drug use in therapy and self-medication, and highlights the endocrine side effects after drug therapy in neurological diseases and brain tumors. What the reader will gain: The reader is introduced to a CYP mediated drug–hormone crosstalk as a possible mechanism to explain these side effects. Take home message: The drug–hormone crosstalk may be of considerable importance in the assessment of neuroactive drugs and future drug design.