2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †.

Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency-approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T-positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

The prodrug DHED selectively delivers 17ß-estradiol to the brain for treating estrogen-responsive disorders.

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17ß-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17ß-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17ß-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

A sensitive aß oligomer assay discriminates Alzheimer's and aged control cerebrospinal fluid.

A hallmark of Alzheimer's disease (AD) brain is the amyloid ß (Aß) plaque, which is comprised of Aß peptides. Multiple lines of evidence suggest that Aß oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aß oligomers over Aß monomers with a limit of detection ∼ 0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aß multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aß oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aß oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aß oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.

Oligomers of beta-amyloid are sequestered into and seed new plaques in the brains of an AD mouse model.

Amyloid plaque deposition in the brain is a hallmark of Alzheimer's disease, but recent evidence indicates that the disease may be primarily caused by soluble amyloid-beta (1-42) (Abeta) oligomers or Abeta-derived diffusible ligands (ADDLs). ADDLs induce cognitive deficits in animal models and are thought to assemble in vitro by a mechanism apart from plaque formation. To investigate the in vivo relationship of ADDLs and plaques, biotin-labeled ADDLs (bADDLs) or amylin oligomers (bAMs) were injected into the hippocampus of hAPP overexpressing mice. The brains were collected 1 or 5 weeks after the last treatment and were processed for immunohistochemistry. Staining of tissue 1 week post-treatment showed bADDLs had diffused throughout the tissue and incorporated into plaques. Additionally, small deposits of thioflavin S-negative bADDLs were observed. At 5 weeks post-treatment, thioflavin S-positive material continued to accumulate around plaques containing bADDLs. Thioflavin S-positive material also accrued around bADDL deposits, implying that bADDLs were capable of seeding new plaques. In contrast, bAMs cleared from the brain and did not accumulate in plaques. Together, these data indicate that ADDLs are able to contribute to in vivo plaque formation in a peptide-specific manner.

Inhibition of calcineurin-mediated endocytosis and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors prevents amyloid beta oligomer-induced synaptic disruption.

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid beta (Abeta) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Abeta oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.

Alzheimer's disease-type neuronal tau hyperphosphorylation induced by A beta oligomers.

Alzheimer's disease (AD) is characterized by presence of extracellular fibrillar A beta in amyloid plaques, intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated tau and elevated brain levels of soluble A beta oligomers (ADDLs). A major question is how these disparate facets of AD pathology are mechanistically related. Here we show that, independent of the presence of fibrils, ADDLs stimulate tau phosphorylation in mature cultures of hippocampal neurons and in neuroblastoma cells at epitopes characteristically hyperphosphorylated in AD. A monoclonal antibody that targets ADDLs blocked their attachment to synaptic binding sites and prevented tau hyperphosphorylation. Tau phosphorylation was blocked by the Src family tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide (PP1), and by the phosphatidylinositol-3-kinase inhibitor LY294002. Significantly, tau hyperphosphorylation was also induced by a soluble aqueous extract containing A beta oligomers from AD brains, but not by an extract from non-AD brains. A beta oligomers have been increasingly implicated as the main neurotoxins in AD, and the current results provide a unifying mechanism in which oligomer activity is directly linked to tau hyperphosphorylation in AD pathology.

Amphetamine-induced locomotor activity is reduced in mice following MPTP treatment but not following selegiline/MPTP treatment.

MPTP treatment has been used in mice to cause dopaminergic neuronal cell loss and subsequent behavioral abnormalities. As such, this animal model is often used as a method for the characterization of putative novel therapeutics for disease states characterized by dopamine loss, such as Parkinson's disease. Previous reports of behavioral abnormalities in mice following MPTP intoxication, however, have been conflicting. For example, open field spontaneous activity has been reported to increase, decrease or not change in MPTP treated mice. Accordingly, a more robust and direct functional measure of MPTP-induced central dopamine depletion is needed. In the present manuscript, we report on the characterization of amphetamine-induced locomotor activity as a sensitive functional endpoint for dopamine loss following MPTP treatment. We found that the amphetamine-induced locomotor activity of C57BL/6 mice was reduced in a dose-dependent manner following treatment with MPTP. This reduction of activity was associated with decreases in central dopamine levels. Further, the potential for use of this endpoint to evaluate putative therapeutics is exemplified by the amelioration of these effects following pre-treatment with the MAO-B inhibitor selegiline.

Differential modulation of estrogen receptors (ERs) in ischemic brain injury: a role for ERalpha in estradiol-mediated protection against delayed cell death.

Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERalpha and ERbeta, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERalpha induction occurring early and ERbeta modulation occurring later. Finally, we provide evidence for a new and functional role for ERalpha in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERalpha.

Estrogen attenuates the MPTP-induced loss of dopamine neurons from the mouse SNc despite a lack of estrogen receptors (ERalpha and ERbeta).

Estrogen attenuates the loss of dopamine from striatum and dopamine neurons from the substantia nigra (SNc) in animal models of Parkinson's disease. Interestingly, estrogen receptors (ERalpha and ERbeta) are thought to be sparse or absent in mouse striatum and SNc. Since ERalpha is markedly induced in rodent cortex after ischemic injury, the present studies evaluated changes in ERs after acute treatment with the dopamine neurotoxin MPTP. Mice were injected daily with estradiol, injected with MPTP on day 6, and brains collected on day 9 or 13. Immunocytochemistry was then used to assess tyrosine hydroxylase (TH) in striatum and investigate the localization of ERalpha and ERbeta in the striatum and SNc. In addition, cryostat sections were hybridized with a riboprobe complementary to ERalpha or ERbeta mRNA. Evaluation of TH immunoreactivity revealed a dense network of fibers in the striatum of vehicle-treated animals, while a near complete loss of terminals was seen after MPTP treatment. When, however, mice were pretreated with estradiol, the MPTP-induced loss of TH was attenuated. Evaluation of ERalpha and ERbeta in the SNc and striatum demonstrated a sparse localization of both ERs in vehicle-treated mice, a pattern that did not change in animals treated with vehicle/MPTP or estradiol/MPTP. These data demonstrate that ERs are sparse in the mouse striatum and SNc and show that this pattern does not change after MPTP intoxication. This observation and the finding that estrogen affords some protection against MPTP suggest that estrogen may act via nuclear receptor independent mechanisms to protect dopamine neurons from toxins such as MPTP.

Localization of B1 bradykinin receptor mRNA in the primate brain and spinal cord: an in situ hybridization study.

The bradykinin 1 and 2 receptors (B1R, B2R) are important mediators of cardiovascular homeostasis, inflammation, and nociception. While B2R is constitutively expressed in many tissues, B1R expression is thought to be absent, but induced under proinflammatory conditions. However, recent data from knockout mice have indicated that B1R acts centrally to mediate nociception, a finding that suggests the constitutive presence of B1R in brain and/or spinal cord. The purpose of the present study was to further elucidate the physiological role of B1R by evaluating the localization of B1R mRNA in the nonhuman primate brain and spinal cord with in situ hybridization. Cryostat sections from monkey brain and spinal cord were hybridized with a [(35)S]-labeled riboprobe complementary to B1R mRNA, stringently washed, and apposed to film and emulsion. The results of these studies revealed the presence of B1R mRNA throughout the rostral-caudal extent of the brain and spinal cord. In particular, labeled cells were seen in the cerebral and entorhinal cortex, dentate gyrus, and pyramidal neurons of the hippocampus, in the thalamus, hypothalamus, amygdala, pontine nuclei, spinal cord, and dorsal root ganglion. Together the present findings offer detailed information about the distribution of B1R mRNA in the primate brain and spinal cord and demonstrate a basal level of expression in the primate nervous system. Moreover, these data provide a foundation for understanding the central actions of kinins and their putative role in mediating a number of processes, including pain and nociception.

DISC1 (Disrupted in Schizophrenia-1) is expressed in limbic regions of the primate brain.

Disrupted in Schizophrenia-1 (DISC1) was identified as truncated by a balanced translocation segregating with schizophrenia and other major mental illness in a large Scottish family. As a step in evaluating the function of DISC1 and its potential role in human schizophrenia, we have determined its regional expression in the primate brain by in situ hybridization. DISC1 expression is highly localized, with most prominent expression in the dentate gyrus of the hippocampus and lateral septum, and lower levels of expression in the cerebral cortex, amygdala, paraventricular hypothalamus, cerebellum, interpeduncular nucleus, and subthalamic nucleus. Given that many of these regions have been implicated in schizophrenia pathogenesis, these results suggest brain circuits through which DISC1 truncation may predispose to schizophrenia.

Neuroprotection by estrogen in animal models of global and focal ischemia.

Estrogen has been demonstrated to protect against brain injury, neurodegeneration, and cognitive decline. Furthermore, estrogen seems to specifically protect cortical and hippocampal neurons from ischemic injury. Here our data evaluating the neuroprotective effects of estrogens, the selective estrogen receptor modulators (SERMs), and estrogen receptor alpha- and beta-selective ligands in animal models of ischemic injury are discussed. In rats and mice, the middle cerebral artery occlusion (MCAO) model was used as models representing cerebrovascular stroke, while in gerbils the two-vessel occlusion model, resenting acute heart attack, was used. Using focal ischemia in ovariectomized ERalphaKO, ERbetaKO, and wild-type mice, we clearly established that the ERalpha subtype is the critical ER-mediating neuroprotection in mouse focal ischemia. Because of the characteristic blood supply of the gerbil, the gerbil global ischemia model was used to evaluate the neuroprotective effects of estrogen, SERMs, and ERalpha- and ERbeta-selective compounds in the hippocampus. Analysis of neurogranin mRNA, a marker of viability of hippocampal neurons, with in situ hybridization, revealed that estrogen treatment resulted in a complete protection in the CA1 regions not only when administered before, but also when given 1 hour after occlusion. Our in vivo binding studies with (125)I-estrogen in gerbils revealed the presence of nuclear estrogen binding sites primarily in CA1 neurons, but not in the CA3 region, as we saw in rats and mice. Together, these observations demonstrate that estrogen protects from ischemic injury in both the focal and global ischemia models by acting primarily via classical nuclear receptors.

Cloning and characterization of Disc1, the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1).

We cloned the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1), a candidate gene for schizophrenia. Disc1 is 3163 nucleotides long and has 60% identity with the human DISC1. Disc1 encodes 851 amino acids and has 56% identity with the human protein. Disc1 maps to the DISC1 syntenic region in the mouse, and genomic structure is conserved. A Disc1 splice variant deletes a portion of Disc1 beginning at amino acids orthologous to the human truncation. Bioinformatic analysis and cross-species comparisons revealed sequence conservation distributed across the genes and conservation of leucine zipper and coiled-coil domains in both orthologs. In situ hybridization in adult mouse brain revealed a restricted expression pattern, with highest levels in the dentate gyrus of the hippocampus and lower expression in CA1-CA3 of the hippocampus, cerebellum, cerebral cortex, and olfactory bulbs. Identification of Disc1 will facilitate the study of DISC1's function and creation of mouse models of DISC1 disruption.

Estrogen modulates oxytocin gene expression in regions of the rat supraoptic and paraventricular nuclei that contain estrogen receptor-beta.

Oxytocin is an important modulator of female reproductive functions including parturition, lactation and maternal behavior, while vasopressin regulates water balance and acts as a neurotransmitter. For decades, it has been suggested that estrogen regulates the production and/or release of oxytocin and vasopressin in the rodent brain. Although several studies demonstrated that estrogen can modulate vasopressin mRNA levels in regions known to contain estrogen receptor (ER), such as the bed nucleus of the stria terminalis and medial amygdala, data from the paraventricular and supraoptic nuclei were inconclusive. Since early immunohistochemical and in situ hybridization studies revealed few, if any, ER containing cells in these hypothalamic nuclei, it was thought that oxytocin and vasopressin were not directly regulated by estrogen. The discovery of a second ER (ER-beta) in the late 1990s suggested that estrogen could act in many brain regions heretofore not considered targets for estrogen action. Initial in situ hybridization studies revealed a wide distribution of ER-beta mRNA in the rat brain including neurons of the supraoptic nucleus and the parvocellular and magnocellular divisions of the paraventricular nucleus. Subsequent double-label in situ hybridization/immunocytochemistry studies showed that ER-beta mRNA was present in oxytocin and vasopressin neurons, with the degree of colocalization being both neuropeptide and region specific. In an attempt to demonstrate that ER-beta mRNA was translated into a biologically active protein, a series of in vivo binding studies were conducted in rats with 125I-estrogen. These data revealed the presence of nuclear estrogen binding sites in neurons of the magnocellular system indicating that ER-beta mRNA was translated into protein. Concurrent studies in mice found that the distribution of ER-beta mRNA and 125I-estrogen binding was similar to rats, although there were some notable differences. For example, ER-beta mRNA and binding were not detected in the mouse supraoptic nucleus and although ER-beta was the principle ER in the paraventricular nucleus, ER-alpha was also present. The prevalence of ERs in the mouse paraventricular nucleus was further investigated using ER-alpha and ER-beta knockout mice for in vivo binding studies with 125I-estrogen. The results of these studies showed that ER-beta was the predominant ER in the paraventricular nucleus and confirmed the presence of ER-beta in other brain regions. Moreover, our group recently generated and characterized several polyclonal antisera raised against the C-terminus of ER-beta. Through the use of these antisera, we have confirmed the presence of ER-beta in the rat paraventricular and supraoptic nuclei and shown that ER-beta is colocalized, in part, with oxytocin and vasopressin. To assess the ability of estrogen to modulate the expression of oxytocin mRNA, ovariectomized rats were treated with vehicle or estradiol and the brains processed for in situ hybridization. The results of these studies revealed that estradiol down-regulated oxytocin mRNA in the rat paraventricular nucleus within 6 h of treatment. Together these data and the observation that some of the oxytocin and vasopressin neurons contain ER-beta suggest that estrogen, acting through ER-beta, may directly regulate oxytocin gene expression. However, since the paraventricular nucleus has many subdivisions with different projections and the degree of colocalization of ER-beta with oxytocin/vasopressin varies among subdivisions, the effects of estrogen treatment on gene expression requires further study to ascertain the role of estrogen action in this neuronal systems.

Methods to enhance signal using isotopic in situ hybridization.

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

Estrogen-binding sites and their functional capacity in estrogen receptor double knockout mouse brain.

Early studies found estrogen-binding sites in the ER knockout (ERalphaKO) mouse brain, suggesting a splice variant of ERalpha or another ER. The discovery of ERbeta suggested that binding was due to ERbeta, although questions about an ERgamma remained. To test this hypothesis, ERbetaKO mice were generated and crossed with ERalphaKO mice, and ERalpha/betaKO animals were used for in vivo binding studies with [(125)I]estrogen. The results revealed nuclear binding sites in the ERalpha/betaKO hypothalamus and amygdala. As the binding resembled the distribution of ERalpha, we evaluated the presence of ERalpha splicing variants. A nonphysiological splice variant of ERalpha was identified in ERalpha/betaKO brain and uterus, but was absent in wild-type mice. ERalpha immunoreactivity was also detected in regions of ERalpha/betaKO brain where residual binding was seen. To ascertain the functionality of the variant, the regulation of PR was assessed in brain. The results revealed that E2 significantly increased PR expression, an indication that the variant can regulate gene transcription. These data demonstrate the presence and functionality of an ERalpha variant in ERalpha/betaKO brain and suggest that the residual binding and regulation of PR in ERalpha/betaKO brain can be accounted for by the variant.

Age differentially influences estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) gene expression in specific regions of the rat brain.

Estradiol's ability to influence neurochemical events that are critical to female reproductive cyclicity and behavior decreases with age. We tested the hypothesis that decreases in estrogen receptor-alpha (ERalpha) and/or ERbeta mRNA explain the brain's declining responsiveness to estradiol. We assessed ERalpha and ERbeta mRNA levels in intact and ovariectomized estradiol-treated rats. ERbeta mRNA was detected in several brain regions and decreased by middle-age in the cerebral cortex and supraoptic nucleus of estradiol-treated rats. ERbeta mRNA levels exhibited a diurnal rhythm in the suprachiasmatic nucleus of young and middle-aged rats and this rhythm was blunted in old rats. We examined ERalpha mRNA in the periventricular preoptic, medial preoptic, ventromedial and arcuate nuclei, and it was decreased only in the periventricular preoptic nucleus of the old rats. In summary, the expression of ERalpha and ERbeta mRNAs is differentially modulated in the aging brain and changes are region specific.