Jeffrey A. Fein

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

A novel member of the opioid receptor family (ORL‐1) has been cloned from a variety of vertebrates. ORL‐1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N‐terminus of ORL‐1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II–V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1–CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL‐1 was observed in telencephalic areas such as caudate‐putamen. In the cerebellum, ORL‐1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL‐1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL‐1 receptor in the rat CNS.

Biochemical markers in persons with preclinical familial Alzheimer disease.

Background: Persons at risk for familial Alzheimer disease (FAD) provide a model in which biomarkers can be studied in presymptomatic disease. Methods: Twenty-one subjects at risk for presenilin-1 (n = 17) or amyloid precursor protein (n = 4) mutations underwent evaluation with the Clinical Dementia Rating (CDR) scale. We obtained plasma from all subjects and CSF from 11. Plasma (Aβ40, Aβ42, F2-isoprostanes) and CSF (F2-isoprostanes, t-tau, p-tau181, Aβ40, Aβ42, and Aβ42/Aβ40 ratio) levels were compared between FAD mutation carriers (MCs) and noncarriers (NCs). Results: Plasma Aβ42 levels (25.1 pM vs 15.5 pM, p = 0.031) and the ratio of Aβ42/Aβ40 (0.16 vs 0.11, p = 0.045) were higher in presymptomatic MCs. Among MCs, those with CDR scores of 0.5 had lower plasma Aβ42 levels than those with CDR scores of 0 (14.1 pM vs 25.1, p = 0.02). The ratio of Aβ42 to Aβ40 was also reduced in the CSF (0.08 vs 0.15, p = 0.046) of nondemented MCs compared to NCs. Total CSF tau and p-tau181 levels were elevated in presymptomatic FAD MCs. CSF levels of F2-isoprostanes were also elevated in MCs (n = 7, 48.6 pg/mL) compared to NCs (n = 4, 21.6 pg/mL, p = 0.031). Conclusions: Our data indicate that Aβ42 is elevated in plasma in familial Alzheimer disease (FAD) mutation carriers (MCs) and suggests that this level may decrease with disease progression prior to the development of overt dementia. We also demonstrated that the ratio of Aβ42 to Aβ40 was reduced in the CSF of nondemented MCs and that elevations of t-tau and p-tau181 are sensitive indicators of presymptomatic disease. Our finding of elevated F2-isoprostane levels in the CSF of preclinical FAD MCs suggests that oxidative stress occurs downstream to mismetabolism of amyloid precursor protein.

Synaptic Changes in Alzheimer’s Disease : Increased Amyloid-β and Gliosis in Surviving Terminals Is Accompanied by Decreased PSD-95 Fluorescence

In an effort to examine changes that precede synapse loss, we have measured amyloid-beta and a series of damage markers in the synaptic compartment of Alzheimers disease (AD) cases. Because localization of events to the terminal region in neurons is problematic with conventional methods, we prepared synaptosomes from samples of cryopreserved human association cortex, and immunolabeled terminals with a procedure for intracellular antigens. Fluorescence was quantified using flow cytometry. The viability dye calcein AM was unchanged in AD terminals compared to controls, and the fraction of large synaptosome particles did not change, although a striking loss of large terminals was observed in some AD cases. The percent positive fraction for a series of pre- and postsynaptic markers was not affected by AD in this cohort. However, the amyloid-beta-positive fraction increased from 16 to 27% (P < 0.02) in terminals from AD cortex. The expression level on a per-terminal basis is indicated in this assay by fluorescence (relative fluorescence units). The fluorescence of presynaptic markers did not change in AD terminals, but PSD-95 fluorescence was decreased by 19% (P < 0.03). Amyloid-beta fluorescence was increased by 132% (P < 0.01), and glial fibrillary acidic protein labeling by 31% (P < 0.01). These results suggest that synapse-associated amyloid-beta is prominent in regions relatively unaffected by AD lesions, and that amyloid accumulation in surviving terminals is accompanied by gliosis and alteration in the postsynaptic structure.

Orphanin FQ inhibits synaptic transmission and long-term potentiation in rat hippocampus.

It is known that opioid peptides acting on opioid receptors can modulate hippocampal synaptic functions. Although a novel member of the opioid receptor family, ORL1 receptors, that displays high‐sequence homology with classical opioid receptors is abundant in the hippocampus, little is known regarding its role in synaptic function. The present study was designed to investigate whether activation of the ORL1 receptor by its natural ligand, orphanin FQ, could modulate synaptic transmission and synaptic plasticity in the hippocampus. The actions of orphanin FQ in the CA1 and dentate gyrus were examined by field potential recordings in response to stimulation of Schaffer collaterals and perforant path, respectively. Our results showed that orphanin FQ, but not the inactive analog des‐Phe1‐orphanin FQ, reduced both the slope of the excitatory postsynaptic potentials and population spike amplitude. The inhibitory effect of orphanin FQ is dose dependent and probably involves a presynaptic mechanism, as suggested by the significantly increased paired‐pulse facilitation evoked in the presence of orphanin FQ. In addition, orphanin FQ was found to inhibit the induction of long‐term potentiation at the Schaffer collateral‐CA1 synapse. These results demonstrate that orphanin FQ can function as an inhibitory modulator regulating synaptic transmission and synaptic plasticity in the hippocampus, suggesting that activation of ORL1 receptors may play an important role in synaptic plasticity involved in learning and memory. Hippocampus 7:88–94, 1997.

Co-localization of amyloid beta and tau pathology in Alzheimer's disease synaptosomes.

The amyloid cascade hypothesis proposes that amyloid beta (Abeta) pathology precedes and induces tau pathology, but the neuropathological connection between these two lesions has not been demonstrated. We examined the regional distribution and co-localization of Abeta and phosphorylated tau (p-tau) in synaptic terminals of Alzheimers disease brains. To quantitatively examine large populations of individual synaptic terminals, flow cytometry was used to analyze synaptosomes prepared from cryopreserved Alzheimers disease tissue. An average 68.4% of synaptic terminals in the Alzheimers disease cohort (n = 11) were positive for Abeta, and 32.3% were positive for p-tau; Abeta and p-tau fluorescence was lowest in cerebellum. In contrast to synaptic p-tau, which was highest in the entorhinal cortex and hippocampus (P = 0.004), synaptic Abeta fluorescence was significantly lower in the entorhinal cortex and hippocampus relative to neocortical regions (P = 0.0003). Synaptic Abeta and p-tau fluorescence was significantly correlated (r = 0.683, P < 0.004), and dual-labeling experiments demonstrated that 24.1% of Abeta-positive terminals were also positive for p-tau, with the highest fraction of dual labeling (39.3%) in the earliest affected region, the entorhinal cortex. Western blotting experiments show a significant correlation between synaptic Abeta levels measured by flow cytometry and oligomeric Abeta species (P < 0.0001). These results showing overlapping Abeta and tau pathology are consistent with a model in which both synaptic loss and dysfunction are linked to a synaptic amyloid cascade within the synaptic compartment.

Increased cholesterol in Aβ-positive nerve terminals from Alzheimer's disease cortex

Synapse loss in Alzheimers disease (AD) is poorly understood but evidence suggests it is a key pathological event. In order to precisely detect stable synaptic changes, we have developed methods for flow cytometry analysis of synaptosomes prepared from cryopreserved AD samples, and have previously shown that amyloid-beta (Abeta) accumulates in surviving presynaptic terminals in AD cortex. In the present experiments we have examined amyloid-containing terminals in more detail, first dual labeling synaptosomes from AD cortex for Abeta and a series of markers, and then using quadrant analysis to compare amyloid-positive and amyloid-negative terminals. Amyloid-positive synaptosomes were larger in size than amyloid-negatives (p<0.007), and significant increases were observed in mean fluorescence for the lipid raft markers cholesterol (27%; p<0.0005) and GM1 ganglioside (24%; p<0.005). SNAP-25 immunofluorescence was increased by 31% (p<0.0001) in amyloid-bearing terminals, consistent with a sprouting response to amyloid accumulation. These results suggest that Abeta accumulation in synaptic terminals may underly dysfunction prior to or independent of extracellular amyloid deposition.

Apolipoprotein E enhances uptake of soluble but not aggregated amyloid‐β protein into synaptic terminals

The cellular mechanism by which apolipoprotein E (apoE) affects the pathogenesis of Alzheimers disease (AD) is not understood. We have examined the effect of apolipoprotein E on the internalization of exogenous amyloid‐β 1–40 (Aβ40) into a rat brain crude synaptosomal preparation. Aβ40 peptide in soluble (within 1 h of dilution in buffer) or aggregated (aged 4 days before dilution in buffer) form was pre‐incubated with lipidated apoE then added to synaptosomes; intraterminal amyloid‐β labeling was quantified using flow cytometry following immunolabeling with the anti‐Aβ (10G4) antibody. The number of Aβ‐positive synaptosomes was increased (∼50%) by treatment with a soluble Aβ/apoE mixture compared with treatment with soluble Aβ40 alone. However, when the Aβ was aggregated, less sodium dodecyl sulfate (SDS)‐stable Aβ/apoE complex was formed and the addition of apoE decreased the number of Aβ‐positive terminals. The addition of the lipoprotein‐receptor related protein (LRP) antagonist receptor‐associated protein (RAP) inhibited the apoE‐induced increase in synaptosomal Aβ, and controls treated with trypsin and heparinase confirm intraterminal localization of the majority of the soluble Aβ. The apoE‐mediated increase in Aβ labeling was confirmed in intact cells by immunocytochemistry of dorsal root ganglion (DRG) neurons. These results suggest that complex formation with apoE enhances internalization of soluble Aβ uptake into terminals.

ORL‐1 and Mu opioid receptor antisera label different fibers in areas involved in pain processing

Mu opioid receptors (MOR) mediate the analgesic effects of opioid drugs such as morphine. The opioid receptor‐like (ORL‐1) receptor is structurally related to opioid receptors and the ORL‐1 receptor agonist, orphanin FQ/nociceptin, induces analgesia at the spinal level, but appears to recruit different circuitry than that used by mu opioids. When administered intracerebroventricularly, orphanin FQ/nociceptin produces hyperalgesia and/or reverses opioid analgesia. The functionally distinct actions elicited by MOR and ORL‐1 receptors, which activate similar intracellular signaling systems and show similar regional distributions, could be explained by their differential cellular localization. By using double label immunohistochemistry and confocal microscopy, the present study investigates the distribution of MOR and ORL‐1 receptors in regions of the rat nervous system that are involved with nociceptive processing. In general co‐localization of MOR and ORL‐1 receptor immunoreactivity was not observed in either perikarya or neuropil in the dorsal root ganglia, nor in the Lissauers tract and superficial laminae of the spinal cord. Likewise, there was no evidence for co‐localization of these receptors within the periaqueductal gray, the nucleus raphe magnus, the gigantocellular reticular nucleus, and the nucleus of the solitary tract. These observations indicate that MOR and ORL‐1 receptors are expressed predominantly on different fiber systems in these regions. This differential distribution is consistent with the distinct pharmacology of ORL‐1 and MOR receptor agonists and suggests that the antisera to MOR and ORL‐1 receptors may provide useful markers for further investigations of analgesic and counteranalgesic pathways modulating pain perception. J. Comp. Neurol. 399:373–383, 1998.

Quantitative characterization of crude synaptosomal fraction (P-2) components by flow cytometry.

Flow cytometry, which definitively identifies each particle as positive or negative with respect to fluorescent markers, is used to characterize the P‐2 fraction (crude synaptosomal fraction) with respect to primary components, size, and intactness. Particle size ranged from a few tenths of a μm to greater than 4.5 μm. The viable dye calcein AM labeled 90% of the preparation, indicating that the majority of particles were intact and esterase‐positive. 66% of the P‐2 fraction is neuronal in origin, as demonstrated by labeling with an antibody directed against SNAP‐2. An antibody directed against glial fibrillary acidic protein (GFAP) labeled 35% of the particles in this preparation. The mitochondrial dye nonyl acridine orange (NAO) stained 74% of particles, indicating intra‐ and extrasynaptosomal mitochondria. Gating analysis reveals that SNAP‐25 is enriched in the larger particles. These results suggest that flow cytometry may be used to take advantage of the increased viability, yield, and convenience of the P‐2 fraction for studies of nerve terminal function. J. Neurosci. Res. 61:186–192, 2000.

Enrichment of presynaptic and postsynaptic markers by size‐based gating analysis of synaptosome preparations from rat and human cortex

Synapse regions in the brain are difficult to isolate and study; resealed nerve terminals (synaptosomes) are a widely used in vitro system for the study of neurotransmission, but nonsynaptosomal elements in the homogenate complicate data interpretation. With the goal of quantitative analysis of pathways leading to synapse loss in neurodegenerative disease, we have developed a method that allows focus on the intact synaptosomes within a crude synaptosomal preparation by gating the largest particles based on forward angle light scatter (FSC).