John F. Ervin

Specific Regional Transcription of Apolipoprotein E in Human Brain Neurons

In central nervous system injury and disease, apolipoprotein E (APOE, gene; apoE, protein) might be involved in neuronal injury and death indirectly through extracellular effects and/or more directly through intracellular effects on neuronal metabolism. Although intracellular effects could clearly be mediated by neuronal uptake of extracellular apoE, recent experiments in injury models in normal rodents and in mice transgenic for the human APOE gene suggest the additional possibility of intraneuronal synthesis. To examine whether APOE might be synthesized by human neurons, we performed in situ hybridization on paraffin-embedded and frozen brain sections from three nondemented controls and five Alzheimers disease (AD) patients using digoxigenin-labeled antisense and sense cRNA probes to human APOE. Using the antisense APOE probes, we found the expected strong hybridization signal in glial cells as well as a generally fainter signal in selected neurons in cerebral cortex and hippocampus. In hippocampus, many APOE mRNA-containing neurons were observed in sectors CA1 to CA4 and the granule cell layer of the dentate gyrus. In these regions, APOE mRNA containing neurons could be observed adjacent to nonhybridizing neurons of the same cell class. APOE mRNA transcription in neurons is regionally specific. In cerebellar cortex, APOE mRNA was seen only in Bergmann glial cells and scattered astrocytes but not in Purkinje cells or granule cell neurons. ApoE immunocytochemical localization in semi-adjacent sections supported the selectivity of APOE transcription. These results demonstrate the expected result that APOE mRNA is transcribed and expressed in glial cells in human brain. The important new finding is that APOE mRNA is also transcribed and expressed in many neurons in frontal cortex and human hippocampus but not in neurons of cerebellar cortex from the same brains. This regionally specific human APOE gene expression suggests that synthesis of apoE might play a role in regional vulnerability of neurons in AD. These results also provide a direct anatomical context for hypotheses proposing a role for apoE isoforms on neuronal cytoskeletal stability and metabolism.

The neurosteroid allopregnanolone is reduced in prefrontal cortex in Alzheimer's disease

BACKGROUND Few data are currently available investigating neurosteroids (NS) in Alzheimers disease (AD). The NS allopregnanolone may be decreased in serum and plasma in patients with AD, but it is unclear if allopregnanolone is also reduced in brain. Because a number of NS exhibit neuroprotective effects and impact cognitive performance in rodent models, these molecules may be relevant to the pathophysiology of neurodegenerative disorders. We therefore investigated prefrontal cortex (PFC) NS levels in AD. METHODS Neurosteroid levels (allopregnanolone, pregnenolone, dehydroepiandrosterone [DHEA]) were determined in postmortem PFC in 14 male subjects with AD and 15 cognitively intact male control subjects by gas chromatography/mass spectrometry preceded by high-performance liquid chromatography purification. RESULTS Subjects with AD exhibit significant reductions in allopregnanolone compared with cognitively intact control subjects (median levels = 2.50 ng/g vs. 5.59 ng/g, respectively; p = .02). Allopregnanolone levels are inversely correlated with neuropathological disease stage (Braak), r = -.49, p = .007. Median DHEA levels are elevated in subjects with AD (p = .01). CONCLUSIONS Subjects with AD demonstrate significant reductions in PFC allopregnanolone levels, a finding that may be relevant to neuropathological disease stage severity. Neurosteroids may have utility as candidate biomarkers in AD.

Reduced levels of human apoE4 protein in an animal model of cognitive impairment

The APOE4 allele is the most common genetic determinant for Alzheimers disease (AD) in the developed world. APOE genotype specific differences in brain apolipoprotein E protein levels have been observed in numerous studies since the discovery of APOE4s link to AD. Since the human apoE4 targeted replacement mice display characteristics of cognitive impairment we sought to determine if reduced levels of apoE might provide one explanation for this impairment. We developed a novel mass spectrometry method to measure apoE protein levels in plasma. Additionally, we developed an ELISA that replicates the mass spectrometry data and enables the rapid quantitation of apoE in plasma, brain and cerebrospinal fluid. We detected a significant decrease in plasma, brain and cerebrospinal fluid apoE levels in the apoE4 mice compared to apoE2 and E3 mice. We also measured a small (∼19%) decrease in brain apoE levels from aged, non-demented APOE4 carriers. Our findings suggest that a fraction of APOE4-linked AD may be due to insufficient levels of functional apoE required to maintain neuronal health.

Cerebrospinal Fluid Dehydroepiandrosterone Levels Are Correlated with Brain Dehydroepiandrosterone Levels, Elevated in Alzheimer’s Disease, and Related to Neuropathological Disease Stage

OBJECTIVE It is currently unknown whether cerebrospinal fluid (CSF) neurosteroid levels are related to brain neurosteroid levels in humans. CSF and brain dehydroepiandrosterone (DHEA) levels are elevated in patients with Alzheimers disease (AD), but it is unclear whether CSF DHEA levels are correlated with brain DHEA levels within the same subject cohort. We therefore determined DHEA and pregnenolone levels in AD patients (n = 25) and cognitively intact control subjects (n = 16) in both CSF and temporal cortex. DESIGN DHEA and pregnenolone levels were determined by gas chromatography/mass spectrometry preceded by HPLC. Frozen CSF and temporal cortex specimens were provided by the Alzheimers Disease Research Center at Duke University Medical Center. Data were analyzed by Mann-Whitney U test statistic and Spearman correlational analyses. RESULTS CSF DHEA levels are positively correlated with temporal cortex DHEA levels (r = 0.59, P < 0.0001) and neuropathological disease stage (Braak and Braak) (r = 0.42, P = 0.007). CSF pregnenolone levels are also positively correlated with temporal cortex pregnenolone levels (r = 0.57, P < 0.0001) and tend to be correlated with neuropathological disease stage (Braak) (r = 0.30, P = 0.06). CSF DHEA levels are elevated (P = 0.032), and pregnenolone levels tend to be elevated (P = 0.10) in patients with AD, compared with cognitively intact control subjects. CONCLUSIONS These findings indicate that CSF DHEA and pregnenolone levels are correlated with temporal cortex brain levels of these neurosteroids and that CSF DHEA is elevated in AD and related to neuropathological disease stage. Neurosteroids may thus be relevant to the pathophysiology of AD.

Mortalin is regulated by APOE in hippocampus of AD patients and by human APOE in TR mice.

Mortalin is a chaperone protein associated with cell survival, stress response, intracellular trafficking, control of cell proliferation, mitochondrial biogenesis, and cell fate determination. Human APOE targeted replacement (TR) mice have been used to elucidate the role of APOE4 in Alzheimers disease (AD), since these animals express the APOE4 gene without the classical pathological signatures of AD. Using proteomics we found that mortalin isoforms are differentially expressed in the hippocampus of APOE4 TR mice compared with the APOE3 (control) TR mice. We also observed that these mortalin isoforms are differentially phosphorylated. Then we studied mortalin expression in patients with AD (genotypes APOE 3/3 and APOE 4/4) compared with patients without AD (genotype APOE 3/3). We observed that mortalin isoforms are also differentially expressed in the hippocampi of patients with AD, and that the expression of these mortalin isoforms is regulated by the APOE genotype. We propose that the differential regulation of mortalin in AD and by the APOE genotype is a cellular defense mechanism responding to increases in oxidative stress.

Postmortem Delay Has Minimal Effect on Brain RNA Integrity

The Bryan Alzheimer Disease Research Center obtains postmortem human brain tissue from patients with Alzheimer disease (AD) and cognitively normal control subjects for molecular and genetic research programs. A growing body of research suggests that variations in gene transcript levels may contribute to the onset and progression of disease. Identifying how the regulation of gene expression may affect AD requires the use of high-quality mRNA from banked human brains. The present study was conducted to establish the quality and suitability of available banked brain tissue for future gene expression studies. We chose 32 AD cases with Braak stage IV, V, or VI. These AD cases were matched to 36 normal control cases by age and sex when possible. Multiple regions from each brain were sampled, including frontal cortex, temporal cortex, occipital cortex, and cerebellum. Hippocampus was also available for study from 14 control cases. A comparison of several antemortem and postmortem variables, such as postmortem interval, agonal state, ventricular cerebrospinal fluid pH, and cause of death were analyzed. RNA was isolated from at least 1 area from every brain and most brains yielded intact RNA from all regions tested. Analysis of the clinical variables did not reveal any features that correlated with the ability to recover intact mRNA. We conclude that undegraded mRNA may be isolated from most brain regions many hours postmortem and that neither the pH of ventricular fluid nor postmortem interval is predictive of mRNA integrity.

Allopregnanolone levels are reduced in temporal cortex in patients with Alzheimer's disease compared to cognitively intact control subjects.

The neurosteroid allopregnanolone has pronounced neuroprotective actions, increases myelination, and enhances neurogenesis. Evidence suggests that allopregnanolone dysregulation may play a role in the pathophysiology of Alzheimers disease (AD) and other neurodegenerative disorders. Our prior data demonstrate that allopregnanolone is reduced in prefrontal cortex in male patients with AD compared to male cognitively intact control subjects, and inversely correlated with neuropathological disease stage (Braak and Braak). We therefore determined if allopregnanolone levels are also reduced in AD patients compared to control subjects in temporal cortex, utilizing a larger set of samples from both male and female patients. In addition, we investigated if neurosteroids are altered in subjects who are APOE4 allele carriers. Allopregnanolone, dehydroepiandrosterone (DHEA), and pregnenolone levels were determined in temporal cortex postmortem samples by gas chromatography/mass spectrometry, preceded by high performance liquid chromatography (40 subjects with AD/41 cognitively intact control subjects). Allopregnanolone levels are reduced in temporal cortex in patients with AD (median 2.68 ng/g, n=40) compared to control subjects (median 5.64 ng/g, n=41), Mann-Whitney p=0.0002, and inversely correlated with Braak and Braak neuropathological disease stage (Spearman r=-0.38, p=0.0004). DHEA and pregnenolone are increased in patients with AD compared to control subjects. Patients carrying an APOE4 allele demonstrate reduced allopregnanolone levels in temporal cortex (Mann-Whitney p=0.04). In summary, our findings indicate that neurosteroids are altered in temporal cortex in patients with AD and related to neuropathological disease stage. In addition, the APOE4 allele is associated with reduced allopregnanolone levels. Neurosteroids may be relevant to the neurobiology and therapeutics of AD.

Lewy body pathology is a frequent co-pathology in familial Alzheimer's disease

Our institution is currently engaged in ongoing genetic studies of familial Alzheimers disease (AD), which include clinical ascertainment and brain autopsy of both affected and non-affected family members. Here we describe the analysis of 22 AD families, each with at least one family member with a postmortem diagnosis of dementia with Lewy bodies (DLB). For this study, 47 brains were examined according to NINCDS-Reagan Institute criteria for the diagnosis of AD. Lewy body pathology was evaluated with α-synuclein immunohistochemistry. Four families, with either one or two autopsies showing Lewy body pathology, demonstrated linkage to 12p. Five families had two or more autopsies with Lewy body pathology, but their linkage status was unknown. The remaining 13 families had one autopsy demonstrating Lewy bodies. These findings suggest that at least one pathological form of DLB may be familial. In some families, the pathological phenotype is identical in all examined affected family members; but in others, there may be several pathologies that coexist. Careful neuropathological examination of affected family members may prove critical for future genetic analysis of AD and DLB.

A SAGE study of apolipoprotein E3/3, E3/4 and E4/4 allele-specific gene expression in hippocampus in Alzheimer disease.

APOE4 allele is a major risk factor for late-onset Alzheimer disease (AD). The mechanism of action of APOE in AD remains unclear. To study the effects of APOE alleles on gene expression in AD, we have analyzed the gene transcription patterns of human hippocampus from APOE3/3, APOE3/4, APOE4/4 AD patients and normal control using Serial Analysis of Gene Expression (SAGE). Using SAGE, we found gene expression patterns in hippocampus of APOE3/4 and APOE4/4 AD patients differ substantially from those of APOE3/3 AD patients. APOE3/4 and APOE4/4 allele expression may activate similar genes or gene pools with associated functions. APOE4 AD alleles activate multiple tumor suppressors, tumor inducers and negative regulator of cell growth or repressors that may lead to increased cell arrest, senescence and apoptosis. In contrast, there is decreased expression of large clusters of genes associated with synaptic plasticity, synaptic vesicle docking and fusing and axonal/neuronal outgrowth. In addition, reduction of neurotransmitter receptors and Ca2+ homeostasis, disruption of multiple signal transduction pathways, loss of cell protection, and perhaps most notably, mitochondrial oxidative phosphorylation/energy metabolism are associated with APOE3/4 and APOE4/4 AD alleles. These findings may help define the mechanisms that APOE4 contribute that increase risk for AD and identify new candidate genes conferring susceptibility to AD.

Cardiovascular Damage in Alzheimer Disease: Autopsy Findings From the Bryan ADRC

Autopsy information on cardiovascular damage was investigated for pathologically confirmed Alzheimer disease (AD) patients (n = 84) and non-AD control patients (n = 60). The 51 relevant items were entered into a grade-of-membership model to describe vascular damage in AD. Five latent groups were identified “I: early-onset AD,” “II: controls, cancer,” “III: controls, extensive atherosclerosis,” “IV: late-onset AD, male,” and “V: late-onset AD, female.” Expectedly, Groups IV and V had elevated APOE ϵ4 frequency. Unexpectedly, there was limited atherosclerosis and frequent myocardial valve and ventricular damage. The findings do not indicate a strong relationship between atherosclerosis and AD, although both are associated with the APOE ϵ4. Instead, autopsy findings of extensive atherosclerosis were associated with possible, not probable or definite AD, and premature death. They are consistent with the hypothesis that brain hypoperfusion contributes to dementia, possibly to AD pathogenesis, and raise the possibility that the APOE allele ϵ4 contributes directly to heart valve and myocardial damage.