Heather S. Ryan

Sleep and circadian abnormalities in a transgenic mouse model of Alzheimer’s disease: A role for cholinergic transmission

The Tg2576 mouse model of Alzheimers disease (AD) exhibits age-dependent amyloid beta (Abeta) deposition in the brain. We studied electroencephalographically defined sleep and the circadian regulation of waking activities in Tg2576 mice to determine whether these animals exhibit sleep abnormalities akin to those in AD. In Tg2576 mice at all ages studied, the circadian period of wheel running rhythms in constant darkness was significantly longer than that of wild type mice. In addition, the increase in electroencephalographic delta (1-4 Hz) power that occurs during non-rapid eye movement sleep after sleep deprivation was blunted in Tg2576 mice relative to controls at all ages studied. Electroencephalographic power during non-rapid eye movement sleep was shifted to higher frequencies in plaque-bearing mice relative to controls. The wake-promoting efficacy of the acetylcholinesterase inhibitor donepezil was lower in plaque-bearing Tg2576 mice than in controls. Sleep abnormalities in Tg2576 mice may be due in part to a cholinergic deficit in these mice. At 22 months of age, two additional deficits emerged in female Tg2576 mice: time of day-dependent modulation of sleep was blunted relative to controls and rapid eye movement sleep as a percentage of time was lower in Tg2576 than in wild type controls. The rapid eye movement sleep deficit in 22 month-old female Tg2576 mice was abolished by brief passive immunization with an N-terminal antibody to Abeta. The Tg2576 model provides a uniquely powerful tool for studies on the pathophysiology of and treatments for sleep deficits and associated cholinergic abnormalities in AD.

Markers in the 15q24 nicotinic receptor subunit gene cluster (CHRNA5-A3-B4) predict severity of nicotine addiction and response to smoking cessation therapy.

Stopping smoking is difficult even with treatment. Many patients prescribed pharmacologic treatments for smoking cessation experience side effects or lack of efficacy. We performed a pharmacogenetic study of the efficacy and tolerability of bupropion and transdermal nicotine (TN), two treatments for smoking cessation. Samples were drawn from two studies. In the first study (Maintenance 1, MT1), 301 smokers received bupropion plus TN for 11 weeks, followed by 14 weeks of placebo or bupropion. In the second study (MT2), 276 smokers received bupropion and TN for 8 weeks. We focused on eight SNPs in the 15q24 region, which contains the genes for the nicotinic cholinergic receptor subunits CHRNA5, CHRNA3, and CHRNB4, and has previously been implicated in nicotine addiction and smoking cessation. Analyses of baseline smoking quantity (SQ) identified an association between SQ and both the functional CHRNA5 SNP rs16969968 (D398N) and the CHRNA3 SNP rs1051730 (Y215Y) in a combined cohort containing MT1 and MT2. An association between SQ and ethnicity was also identified in the combined cohort. Pharmacogenetic analysis showed a significant association between rs8192475 (R37H) in CHRNA3 and both higher craving after quitting and increased withdrawal symptoms over time in MT2. Two markers for point prevalence abstinence, CHRNA5 SNP rs680244 and CHRNB4 SNP rs12914008, were also identified in MT2, with the strongest findings at week 52. These results provide further support for the role of the CHRNA5/A3/B4 subunits in determining number of cigarettes smoked and response to smoking cessation therapy.

Analysis of neuronal gene expression with laser capture microdissection

The brain is a heterogeneous tissue in which the numbers of neurons, glia, and other cell types vary among anatomic regions. Gene expression studies performed on brain homogenates yield results reflecting mRNA abundance in a mixture of cell types. Therefore, a method for quantifying gene expression in individual cell populations would be useful. Laser capture microdissection (LCM) is a new technique for obtaining pure populations of cells from heterogeneous tissues. Most studies thus far have used LCM to detect DNA sequences. We developed a method to quantify gene expression in hippocampal neurons from mouse brain using LCM and real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). This method was optimized to permit histochemical or immunocytochemical visualization of nerve cells during LCM while minimizing RNA degradation. As an example, gene expression was quantified in hippocampal neurons from the Tg2576 mouse model for Alzheimers disease.

ABCB1 (MDR1) polymorphisms and antidepressant response in geriatric depression.

Objective Variation in the ATP-binding cassette, subfamily B, member 1 transporter (ABCB1) (multidrug-resistance gene 1) gene has been investigated as a predictor of response to treatment with a variety of medications such as antiarrhythmics, chemotherapeutic agents, anti-HIV medications, and some psychotropics. The ABCB1 gene product, P-glycoprotein, affects the transport of drugs out of many cell types, including endothelial cells at the blood–brain barrier. We sought to determine if ABCB1 polymorphisms predict response to antidepressant treatment in geriatric patients. Methods We compared the effects of ABCB1 genetic variation on the therapeutic response to paroxetine, a P-glycoprotein substrate, and to mirtazapine, which is not thought to be transported by ABCB1, in a sample of 246 elderly patients with major depression treated in a clinical trial setting. A total of 15 single nucleotide polymorphisms in the ABCB1 gene were assessed in each patient. Two of these ABCB1 single nucleotide polymorphisms were earlier reported to predict treatment response in patients prescribed with P-glycoprotein substrate antidepressants. Results The two earlier identified ABCB1 markers for antidepressant response predicted time to remissionin our paroxetine-treated patients, but not in the mirtazapine-treated patients. These results replicate the published findings of others. If a Bonferroni correction for type I error is made, our results do not reach the criteria for statistical significance. However, the Bonferroni correction may be too conservative given the strong linkage disequilibrium among some of the markers and our aim to replicate the earlier published findings. Conclusion Our study provides confirmation that certain ABCB1 polymorphisms predict response to substrate medications in geriatric patients.

Microglia Overexpressing the Macrophage Colony-Stimulating Factor Receptor Are Neuroprotective in a Microglial-Hippocampal Organotypic Coculture System

Microglia with increased expression of the macrophage colony-stimulating factor receptor (M-CSFR; c-fms) are found surrounding plaques in Alzheimers disease (AD) and in mouse models for AD and after ischemic or traumatic brain injury. Increased expression of M-CSFR causes microglia to adopt an activated state that results in proliferation, release of cytokines, and enhanced phagocytosis. To determine whether M-CSFR-induced microglial activation affects neuronal survival, we assembled a coculture system consisting of BV-2 microglia transfected to overexpress the M-CSFR and hippocampal organotypic slices treated with NMDA. Twenty-four hours after assembly of the coculture, microglia overexpressing M-CSFR proliferated at a higher rate than nontransfected control cells and exhibited enhanced migration toward NMDA-injured hippocampal cultures. Surprisingly, coculture with c-fms-transfected microglia resulted in a dramatic reduction in NMDA-induced neurotoxicity. Similar results were observed when cocultures were treated with the teratogen cyclophosphamide. Biolistic overexpression of M-CSFR on microglia endogenous to the organotypic culture also rescued neurons from excitotoxicity. Furthermore, c-fms-transfected microglia increased neuronal expression of macrophage colony-stimulating factor (M-CSF), the M-CSFR, and neurotrophin receptors in the NMDA-treated slices, as determined with laser capture microdissection. In the coculture system, direct contact between the exogenous microglia and the slice was necessary for neuroprotection. Finally, blocking expression of the M-CSF ligand by exogenous c-fms-transfected microglia with a hammerhead ribozyme compromised their neuroprotective properties. These results demonstrate a protective role for microglia overexpressing M-CSFR in our coculture system and suggest under certain circumstances, activated microglia can help rather than harm neurons subjected to excitotoxic and teratogen-induced injury.

FKBP5 polymorphisms and antidepressant response in geriatric depression.

Genetic variation at the FKBP5 locus has been reported to affect clinical outcomes in patients treated with antidepressant medications in several studies. However, other reports have not confirmed this association. FKBP5 may regulate the sensitivity of the hypothalamic–pituitary–adrenal axis. We tested two FKBP5 single nucleotide polymorphisms (rs1360780 and rs3800373) in a sample of 246 geriatric patients treated for 8 weeks in a double‐blind randomized comparison trial of paroxetine and mirtazapine. These two polymorphisms had previously been reported to predict efficacy in depressed patients treated with selective serotonin reuptake inhibitors such as paroxetine, and those treated with mirtazapine, an agent with both serotonergic and noradrenergic actions. However, we found no significant associations between these FKBP5 genetic variants and clinical outcomes. Neither mean Hamilton Depression Rating Scale scores nor time to remission or response were predicted by FKBP5 genetic variation. These results suggest that FKBP5 is unlikely to play a major role in determining antidepressant treatment outcomes in geriatric patients.

Aβ peptide conformation determines uptake and interleukin-1α expression by primary microglial cells

Microglia clear amyloid beta (Abeta) after immunization. The interaction of Abeta with the microglial cell surface also results in cytokine expression. Soluble oligomers and protofibrils of Abeta may be more neurotoxic than Abeta fibrils. We investigated the effects of oligomeric, protofibrillar and fibrillar Abeta40 and Abeta42 peptides on uptake and IL-1alpha expression by primary microglia. Abeta peptide assemblies were extensively characterized. Primary microglial cells were exposed to different Abeta40 and Abeta42 assemblies and IL-1alpha expression was quantified. To study uptake, microglial cells were exposed to different assemblies of Cy3-labeled Abeta. We found that Abeta42 and Abeta40 oligomers and fibrils induced IL-1alpha expression, but protofibrils did not. We also observed that all forms of Abeta42 (oligomer, protofibril and fibril) and Abeta40 fibrils were taken up by the microglial cells. These results demonstrate that microglial cells can take up non-fibrillar Abeta and that oligomeric peptide induces an inflammatory response. The uptake of oligomeric and protofibrillar Abeta by microglia merits further investigation as a potential means for removing these neurotoxic species from the brain.

BDNF and CREB1 genetic variants interact to affect antidepressant treatment outcomes in geriatric depression.

Aim Brain-derived neurotrophic factor (BDNF) is associated with antidepressant response on the cellular level, in animal models, and in clinical studies. A common variant in the BDNF gene results in a substitution of a methionine (Met) for a valine at the amino acid position 66. Previous studies reported that the Met variant results in enhanced response to antidepressant medications. These findings may be at odds with studies indicating that on a cellular level the Met variant impairs the secretion of BDNF. Materials and methods We examined the effects of BDNF single nucleotide polymorphisms (SNPs) in response to the antidepressants paroxetine and mirtazapine in a sample of 246 geriatric patients with major depression, treated in a double-blind, randomized, 8-week clinical trial. We also examined the effects of genetic variation at the BDNF-related loci neurotrophic tyrosine kinase receptor 2, cyclic AMP responsive element binding protein 1 (CREB1), and CREB binding protein. A total of 53 SNPs were genotyped. Results BDNF genetic variation had a significant effect on the efficacy of paroxetine, with patients carrying the Met allele showing impaired response. SNPs at the CREB1 locus, which encodes a transcription factor important in BDNF signaling, also predicted response to paroxetine. Furthermore, we found a significant gene–gene interaction between BDNF and CREB1 that affected response to paroxetine. Because BDNF has been associated with cognitive function, we tested the effects of BDNF SNPs on change in a wide variety of cognitive tests over the 8-week trial, but there were no significant effects of genotype on cognition. Conclusion These results provide new evidence for the importance of the BDNF pathway in antidepressant response in geriatric patients. The negative effect of the Met66 allele on antidepressant outcomes is consistent with basic science findings indicating a negative effect of this variant on BDNF activity in the brain. Further, the effect of BDNF genetic variation on antidepressant treatment is modified by variation in the gene encoding the downstream effector CREB1.

Gene expression profile of the PDAPP mouse model for Alzheimer's disease with and without Apolipoprotein E ,

The APOE epsilon 4 allele is a strong risk factor for Alzheimers disease (AD). However, the molecular basis for this effect remains unclear. We examined expression of approximately 12,000 genes and expressed sequence tags in the hippocampus and cortex of PDAPP (APP(V717)) mice modeling AD that show extensive amyloid beta (A beta) deposition, and in PDAPP mice lacking murine APOE expression, which show marked attenuation of A beta deposition in the brain. Wild type and APOE knockout animals were also examined. Expression levels were determined at the initial stage of A beta deposition, as well as in older animals showing extensive neuropathological changes. Fifty-four transcripts were identified using our statistical analysis as differentially regulated between the PDAPP and PDAPP/APOE ko mice, whereas 31 transcripts were classified as differentially regulated among PDAPP mice and WT animals, and seven transcripts were identified as regulated between the PDAPP/APOE ko animals and the APOE ko animals. Interestingly, many of the differentially regulated genes we detected can be related to biological processes previously shown to be important in AD pathophysiology, including inflammation, calcium homeostasis, cholesterol transport and uptake, kinases and phosphatases involved in tau phosphorylation and dephosphorylation, mitochondrial energy metabolism, protein degradation, neuronal growth, endoplasmic reticulum (ER) stress related proteins, antioxidant activity, cytoskeletal organization, and presenilin binding proteins. Regulated genes also included some not directly associated with AD in the past but likely to be involved in known AD pathophysiologic mechanisms, and others that may represent completely novel factors in the pathogenesis of AD. These results provide a global molecular profile of hippocampal and cortical gene expression during the initial and intermediate stages Abeta deposition, and the effects of APOE deletion on this process.

ABCB1 (MDR1) predicts remission on P-gp substrates in chronic depression

The hypothesis that allelic variation in the multidrug resistance-1 (MDR1 or ABCB1) gene encoding the P-glycoprotein (P-gp) blood–brain barrier efflux pump is associated with remission and side effects was tested in chronic major depression patients treated with P-gp substrates. In 83 patients from the REVAMP trial, frequency of and time to remission as well as side effects was tested among genotype groups at 6 ABCB1 single nucleotide polymorphisms (SNPs). These six SNPs are significantly associated with remission and time to remission, with minor allele carriers on rs2235040 and rs9282564 attaining statistical significance after controlling for the other ABCB1 SNPs. The six ABCB1 SNPs are also significantly associated with the average side effects. However, here common homozygotes on rs2235040 and rs9282564 demonstrated significantly higher side effects after controlling for the effects of the other ABCB1 SNPs. These findings confirm and extend previous observations that minor alleles of two ABCB1 SNPs predict remission to treatment with substrates and demonstrate that common homozygotes on these SNPs experience greater side effects. Results point to the potential importance of ABCB1 variation for personalized medicine approaches to treating depression.