Sally Schroeter

Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter.

Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585–602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine‐ and dopamine‐containing neurons. Intracerebroventricular anti‐dopamine β‐hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP‐25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states. J. Comp. Neurol. 420:211–232, 2000.

Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins

Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) mediate antidepressant-sensitive clearance of 5-HT following release. Although we have been aware for decades that SERT-mediated 5-HT clearance can be modulated by exogenous agents including serotonin-selective reuptake inhibitors, amphetamines, and cocaine, we have had little reason to speculate that SERT activity was actively controlled through endogenous pathways. Recent studies indicate that SERTs are likely to be trafficked to specific plasma membrane subdomains to achieve localized clearance of 5-HT, and that the number of SERTs resident in the plasma membrane is controlled through kinase- and phosphatase-linked pathways. In particular, roles for protein kinase C and phosphatase 2A become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter redistribution and loss of functional uptake capacity. Based on our findings, and the studies of others in native tissues and transfected cells, we propose a model whereby SERTs are organized in a macromolecular complex in the plasma membrane that may serve to locate reuptake activity near release sites. Although many elements of this model remain hypothetical, our findings suggest a much more dynamic picture of transporter-mediated 5-HT reuptake than typically described and suggest opportunities both for the development of new SERT regulatory agents and for the identification of regulatory pathways that may be compromised in mental illness.

Immunotherapy Reduces Vascular Amyloid-β in PDAPP Mice

In addition to parenchymal amyloid-β (Aβ) plaques, Alzheimers disease (AD) is characterized by Aβ in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in the majority of patients. Recent studies investigating vascular Aβ (VAβ) in amyloid precursor protein transgenic mice have suggested that passive immunization with anti-Aβ antibodies may clear parenchymal amyloid but increase VAβ and the incidence of microhemorrhage. However, the influences of antibody specificity and exposure levels on VAβ and microhemorrhage rates have not been well established, nor has any clear causal relationship been identified. This report examines the effects of chronic, passive immunization on VAβ and microhemorrhage in PDAPP mice by comparing antibodies with different Aβ epitopes (3D6, Aβ1–5; 266, Aβ16–23) and performing a 3D6 dose–response study. VAβ and microhemorrhage were assessed using concomitant Aβ immunohistochemistry and hemosiderin detection. 3D6 prevented or cleared VAβ in a dose-dependent manner, whereas 266 was without effect. Essentially complete absence of VAβ was observed at the highest 3D6 dose, whereas altered morphology suggestive of ongoing clearance was seen at lower doses. The incidence of microhemorrhage was increased in the high-dose 3D6 group and limited to focal, perivascular sites. These colocalized with Aβ deposits having altered morphology and apparent clearance in the lower-dose 3D6 group. Our results suggest that passive immunization can reduce VAβ levels, and modulating antibody dose can significantly mitigate the incidence of microhemorrhage while still preventing or reducing VAβ. These observations raise the possibility that Aβ immunotherapy can potentially slow or halt the course of CAA development in AD that is implicated in vascular dysfunction.

Polarized Expression of the Antidepressant-Sensitive Serotonin Transporter in Epinephrine-Synthesizing Chromaffin Cells of the Rat Adrenal Gland ☆

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporters membrane localization in vivo are lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2 termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intra-cellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.

Regulation of Antidepressant-Sensitive Serotonin Transporters

The indoleamine serotonin (5-hydroxytryptamine [5HT]) plays an important role as a chemical messenger in the adult central nervous system (CNS) and periphery, regulating a host of diverse processes, including mood, sleep, sexual drive, gastrointestinal motility, thyroid function, and vasoconstriction (38,55). Though more than a half dozen distinct 5HT receptor subtypes exist to confer the specific actions of 5HT on target cells (45), a single gene product (11,51,71,102) encoding the serotonin transporter (SERT), appears to be exclusively responsible for the inactivation of extracellular 5HT. SERTs are members of a large gene family of Na+ and Cl− coupled, plasma membrane transporters (1,10),most closely related in sequence to the 1-norepinephrine (NE) transporter (NET) ((73,96) and the dopamine (DA) transporter (DAT) (44,60,113,119). SERT expression in the adult CNS appears to be predominantly, if not exclusively, neuronal (5,11,20,100), restricted to serotonergic neurons of the raphe complex and their projections. Clearance of synaptic and extrasynaptic 5HT appears to be the principal role for SERTs; however, certain cells, notably platelets (100,116), utilize SERTs to acquire 5HT from the environment for subsequent release, since they lack the essential biosynthetic machinery for 5HT synthesis. SERTs have also been identified in intestinal crypt epithelia (121), adrenal chromaffin cells (13,118), mast cells (51), medullary thyroid carcinoma cells (21), thyroid follicular cells (117), and placenta (8).

Drug targets in the embryo. Studies on the cocaine- and antidepressant-sensitive serotonin transporter.

A central goal in developmental biology is the elucidation of signaling events that govern the formation of specific tissues and organs during embryonic morphogenesis. The many processes of morphogenesis, including cell proliferation, cell migration, alterations in cell shape, and cell death’ are correlated with the accurate execution of intrinsic genetic programs, which may be triggered and modified by concentration-dependent effects of extrinsic diffusible substances called morphog e n ~ . ~ ~ For example, a gradient of bicoid protein, set up by diffusion during the early syncitial stage of development in Drosophila, advances the determination of cell fate along the anteroposterior axis.5 Well-studied morphogen candidates include retinoic acid,6 hormones,2 and substances that mimic in vivo signals in in vitro assays such as the induction of mesoderm by activin and basic fibroblast growth factor.’ Recently, increasing attention has been given to the role that neurotransmitters, classically defined by their actions in the adult, may play in embryonic development.2 Even though molecules that are spatially and temporally correlated with morphogenetic events may regulate morphogenesis, it is important not only to identify the biochemical nature of the inducing agent, but also to identify how the local competence of responding cells might be regulated during morphogenesis. In the vertebrate embryo, morphogenetic patterns are most often organized not by a simple cascade of inducing signals, but by a spatially and temporally restricted competence of cells to respond to such gradients. An example is the Wnt gene products in Xenopus laevis embryos, which can locally restrict as well as modify the competence of cells to respond to more uniformly distributed inductive signals.8 Our laboratory studies transporter proteins that are both prominent drug targets as well as agents that normally limit the temporal and spatial influence of neurotransmitters raising questions as to their contributions to embryonic drug effects and normal developmental patterning. Below we discuss our initial efforts to characterize murine serotonin transporter cDNAs and expression as a path to understanding its contributions to morphogenesis.

NPT088 reduces both amyloid-β and tau pathologies in transgenic mice

Alzheimers disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid‐β (Aβ) and hyper‐phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein (“NPT088”) consisting of the active fragment of g3p and human‐IgG1‐Fc.

Discovery, preclinical development, and clinical trial approach for npt088, a general amyloid interaction motif (GAIM)-immunoglobulin fusion

identification of biomarkers predicting the risk of developing AD in asymptomatic subjects could provide key insights into the biology and permit risk stratification and targeted preventive intervention in the early stages of AD, prior to onset of clinical symptoms. We used a metabolomics based approach to identify plasma biomarkers potentially associated with risk of developing dementia. Methods: 2067 dementia-free participants from the Framingham Offspring study (mean age1⁄455.969.7 years; 52.4% women) had comprehensive blood metabolite assessment on sera stored at their 5 examination (1991-1995) using liquid chromatography – mass spectrometry (LC-MS) and have also been prospectively assessed for incident dementia (mean follow-up 1⁄4 15.665.2 years). We used multivariate Cox models to relate log-transformed levels of 217metabolites to dementia occurrence, adjusting for age, sex, APOEε4, education and plasma homocysteine. A metabolite set enrichment analysis (MSEA) was also performed using Metaboanalyst 3.0 including all metabolites with a p-value 0.1. Results:A total of 93 participants developed incident dementia. After correction for multiple testing, only plasma anthranilic acid levels were significantly associated with risk of incident dementia (HR1⁄41.40; 95% CI1⁄4[1.15-1.70] per standard deviation increase in level; p1⁄48.08310). Three additional biologically plausible metabolites reached nominal significance: glutamic acid (HR1⁄41.38; 95% CI1⁄4[1.11-1.72]; p1⁄43.80310), taurine (HR1⁄40.74; 95% CI1⁄4[0.60-0.92]; p1⁄46.91310) and hypoxanthine (HR1⁄40.74; 95% CI1⁄4[0.60-0.92]; p1⁄46.93310). Using a set a 28 metabolites with a p-value 0.1, we observed a nominally significant enrichment for metabolites related to stroke (p1⁄42.77310). Conclusions: We have identified four candidate plasma biomarkers for dementia, each of which has been previously associated with dementia risk in small cerebrospinal fluid and nuclear magnetic resonance metabolomics studies. The MSEA adds to the evidence for an important vascular contribution to dementia and AD. We are now attempting to replicate these results through collaborations and study of various endophenotypes.

REDUCTION OF β-AMYLOID AND PHOSPHO-TAU IN TRANSGENIC MICE BY A NOVEL FUSION PROTEIN BIVALENT FOR A GENERAL AMYLOID INTERACTION MOTIF (GAIM)

for the development of both symptom modifying and disease modifying therapeutics. Tau oligomers are toxic to neurons, inhibit long term potentiation in hippocampal slices, impair the formation of associative fear memory in mice, and induce the propagation of tau pathology during disease progression. Tau oligomer specific antibody has been used in a mousemodel of tauopathy to reduce the propagation of tau pathology and improve motor impairment and cognitive deficits validating tau oligomers as a target for drug development. Methods: A highly diverse library of 100,000 druglike small molecules was screened using AlphaLISA for detection of tau self-association, hits were validated and dose response and neurocytotoxicity assays were performed. Medicinal chemistry analysis was used to select hits for secondary assays. Stably transfected cell lines were established for tau oligomer formation assays. Acute toxicity of selected tool compounds was assessed in wild type mice. Proof-of-concept studies in the JNPL3 mouse model (Taconic) were initiated to demonstrate target engagement in vivo and to validate the screening approach. Results: About 60 druglike molecules predicted to have good CNS penetration were selected from 11 chemical series and 19 singletons. The most active hits of 8 series were chosen as tool compounds and for lead development. Some of these compounds showed good dose response in the cell assay. The acute toxicity evaluation showed no effect on the wild type mice at high, medium and low doses. Studies of tau oligomer and larger aggregate formation in the JNPL3 mice are ongoing. Conclusions: Small molecule inhibitors of tau oligomer formation have been advanced to animal studies. This presentation focuses on the progress of the in vivo studies.

IMMUNOGLOBULIN-GENERAL AMYLOID INTERACTION MOTIF (IG-GAIM) MOLECULES TARGET BETA AMYLOID AND NEUROFIBRILLARY TANGLES IN VITRO AND IN VIVO

antioxidants (GSH and catalase activities) were assessed. Furthermore, the Kaplan-Meier test was used to calculate the average life expectancy. Results: 3xTgAD mice showed lower values of macrophage functions, lymphocyte chemotaxis, NK cells proportion and activity, and GSH from the onset of illness, whereas lymphoproliferative response was exacerbated. Moreover, GSSG/GSH ratio and xanthine oxidase activity are higher in these animals during the establishment of the disease. The 3xTgAD mice showed a shorter lifespan (p<0,05) than NTg. Conclusions: The impaired innate immune functions as well as the higher levels of OS in leucocytes seem to show a premature immunosenescence in 3xTgAD at the age when the amyloid plaques begin to appear (4 months). In contrast, the specific lymphocyte functions showed an over-activation. Although, the differences with NTg lessen as the age increases, the premature immune impairment of 3xTgAD supports the higher mortality shown in this model.