In vivo effects of the alpha-synuclein misfolding inhibitor minzasolmin supports clinical development in Parkinson's disease.

Direct targeting of alpha-synuclein (ASYN) has emerged as a disease-modifying strategy for Parkinson's disease and other synucleinopathies which is being approached using both small molecule compounds and ASYN-targeted biologics. Minzasolmin (UCB0599) is an orally bioavailable and brain-penetrant small molecule ASYN misfolding inhibitor in clinical development as a disease-modifying therapeutic for Parkinson's disease. Herein the results of preclinical evaluations of minzasolmin that formed the basis for subsequent clinical development are described. Pharmacokinetic evaluations of intraperitoneal 1 and 5 mg/kg minzasolmin in wildtype mice revealed parallel and dose-proportional exposures in brain and plasma. Three-month administration studies in the Line 61 transgenic mouse model of PD were conducted to measure ASYN pathology and other PD-relevant endpoints including markers of CNS inflammation, striatal DAT labeling and gait. Reductions in ASYN pathology were correlated with improved aspects of gait and balance, reductions in CNS inflammation marker abundance, and normalized striatal DAT levels. These findings provide support for human dose determinations and have informed the translational strategy for clinical trial design and biomarker selection for the ongoing clinical studies of minzasolmin in patients living with early-stage Parkinson's disease (ClinicalTrials.gov ID: NCT04658186; EudraCT Number 2020-003265).

NPT1220-312, a TLR2/TLR9 Small Molecule Antagonist, Inhibits Pro-Inflammatory Signaling, Cytokine Release, and NLRP3 Inflammasome Activation.

Toll-like receptors (TLRs) play a critical role in innate immune system responses to damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). A growing body of evidence suggests that excessive TLR-mediated innate immune system activation can lead to neuronal damage and precipitate or perpetuate neurodegenerative diseases. Among TLR subtypes, both TLR2 and TLR9 have been implicated in neurodegenerative disorders with increased expression of these receptors in the central nervous system being associated with pro-inflammatory signaling and increased burdens of pathologic aggregated proteins. In the current study, we characterized the actions of a combined TLR2/TLR9 antagonist, NPT1220-312, on pro-inflammatory signaling and cytokine release in monocyte/macrophage-derived heterologous cells, human microglia, and murine and human whole blood. NPT1220-312 potently blocked TLR2- and TLR9-mediated release of inflammatory cytokines in monocyte/macrophage cells and in human microglia. NPT1220-312 also blocked TLR2-mediated activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome including IL-1ß, IL-18, and apoptosis-associated speck-like protein containing a CARD (ASC) release to the culture medium of human differentiated macrophages. The ability of NPT1220-312 to inhibit TLR2 mediated pro-inflammatory release of chemokines and cytokines in situ was demonstrated using murine and human whole blood. Together, these findings suggest that blockade of TLR2 and TLR9 may reduce inappropriate production of pro-inflammatory cytokines and chemokines from peripheral and central immune cells and thus potentially provide therapeutic benefit in neuroinflammatory/neurodegenerative disorders.

NPT520-34 improves neuropathology and motor deficits in a transgenic mouse model of Parkinson's disease.

NPT520-34 is a clinical stage, small molecule being developed for the treatment of Parkinson's disease and other neurodegenerative disorders. The therapeutic potential of NPT520-34 was first suggested by findings from cell-based assays of alpha-synuclein clearance. As reported here, NPT520-34 was subsequently evaluated for therapeutically relevant actions in a transgenic animal model of Parkinson's disease that overexpresses human alpha-synuclein and in an acute lipopolysaccharide-challenge model using wild-type mice. Daily administration of NPT520-34 to mThy1-alpha-synuclein (Line 61) transgenic mice for 1 or 3 months resulted in reduced alpha-synuclein pathology, reduced expression of markers of neuroinflammation, and improvements in multiple indices of motor function. In a lipopolysaccharide-challenge model using wild-type mice, a single dose of NPT520-34 reduced lipopolysaccharide-evoked increases in the expression of several pro-inflammatory cytokines in plasma. These findings demonstrate the beneficial effects of NPT520-34 on both inflammation and protein-pathology end points, with consequent improvements in motor function in an animal model of Parkinson's disease. These findings further indicate that NPT520-34 may have two complementary actions: (i) to increase the clearance of neurotoxic protein aggregates; and (ii) to directly attenuate inflammation. NPT520-34 treatment may thereby address two of the predominate underlying pathophysiological aspects of neurodegenerative disorders such as Parkinson's disease.

Structure based design and synthesis of novel Toll-like Receptor 2 (TLR 2) lipid antagonists.

Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable for > 1.0 h in both gastric juice and PBS buffer and could be used as research tools.

The small molecule alpha-synuclein misfolding inhibitor, NPT200-11, produces multiple benefits in an animal model of Parkinson's disease.

Accumulation of alpha-synuclein (ASYN) in neurons and other CNS cell types may contribute to the underlying pathology of synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Multiple Systems Atrophy (MSA). In support of this hypothesis for PD, ASYN immunopositive aggregates are a prominent pathological feature of PD, and mutations and gene multiplications of human wild type (WT) ASYN cause rare familial autosomal-dominant forms of PD. Targeted therapeutics that reduce the accumulation of ASYN could prevent or slow the neurodegenerative processes in PD and other synucleinopathies. NPT200-11 is a novel small molecule inhibitor of ASYN misfolding and aggregation. The effects of NPT200-11 on ASYN neuropathology were evaluated in animal models over expressing human alpha synuclein. Longitudinal studies using retinal imaging in mice expressing a hASYN::GFP fusion protein revealed that 2 months of once daily administration of NPT200-11 (5 mg/kg IP) resulted in a time-dependent and progressive reduction in retinal ASYN pathology. The effects of NPT200-11 on ASYN pathology in cerebral cortex and on other disease-relevant endpoints was evaluated in the Line 61 transgenic mouse model overexpressing human wild type ASYN. Results from these studies demonstrated that NPT200-11 reduced alpha-synuclein pathology in cortex, reduced associated neuroinflammation (astrogliosis), normalized striatal levels of the dopamine transporter (DAT) and improved motor function. To gain insight into the relationship between dose, exposure, and therapeutic benefit pharmacokinetic studies were also conducted in mice. These studies demonstrated that NPT200-11 is orally bioavailable and brain penetrating and established target plasma and brain exposures for future studies of potential therapeutic benefit.

A de novo compound targeting α-synuclein improves deficits in models of Parkinson's disease.

Abnormal accumulation and propagation of the neuronal protein α-synuclein has been hypothesized to underlie the pathogenesis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Here we report a de novo-developed compound (NPT100-18A) that reduces α-synuclein toxicity through a novel mechanism that involves displacing α-synuclein from the membrane. This compound interacts with a domain in the C-terminus of α-synuclein. The E83R mutation reduces the compound interaction with the 80-90 amino acid region of α-synuclein and prevents the effects of NPT100-18A. In vitro studies showed that NPT100-18A reduced the formation of wild-type α-synuclein oligomers in membranes, reduced the neuronal accumulation of α-synuclein, and decreased markers of cell toxicity. In vivo studies were conducted in three different α-synuclein transgenic rodent models. Treatment with NPT100-18A ameliorated motor deficits in mThy1 wild-type α-synuclein transgenic mice in a dose-dependent manner at two independent institutions. Neuropathological examination showed that NPT100-18A decreased the accumulation of proteinase K-resistant α-synuclein aggregates in the CNS and was accompanied by the normalization of neuronal and inflammatory markers. These results were confirmed in a mutant line of α-synuclein transgenic mice that is prone to generate oligomers. In vivo imaging studies of α-synuclein-GFP transgenic mice using two-photon microscopy showed that NPT100-18A reduced the cortical synaptic accumulation of α-synuclein within 1 h post-administration. Taken together, these studies support the notion that altering the interaction of α-synuclein with the membrane might be a feasible therapeutic approach for developing new disease-modifying treatments of Parkinson's disease and other synucleinopathies.

Longitudinal live imaging of retinal α-synuclein::GFP deposits in a transgenic mouse model of Parkinson's Disease/Dementia with Lewy Bodies.

Abnormal α-synuclein (α-syn) accumulation in the CNS may underlie neuronal cell and synaptic dysfunction leading to motor and cognitive deficits in synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Multiple groups demonstrated α-syn accumulation in CNS accessory structures, including the eyes and olfactory terminals, as well as in peripheral organs of Parkinsonian patients. Retinal imaging studies of mice overexpressing fused α-syn::GFP were conducted to evaluate the presence and progression of retinal pathology in a PD/DLB transgenic mouse model. Bright-field image retinal maps and fluorescent images were acquired at 1-month intervals for 3 months. Retinal imaging revealed the accumulation of GFP-tagged α-syn in retinal ganglion cell layer and in the edges of arterial blood vessels in the transgenic mice. Double labeling studies confirmed that the α-syn::GFP-positive cells were retinal ganglion cells containing α-syn. Accumulation of α-syn persisted in the same cells and increased with age. Accumulation of α-syn::GFP was reduced by immunization with single chain antibodies against α-syn. In conclusion, longitudinal live imaging of the retina in the PDGF-α-syn::GFP mice might represent a useful, non-invasive tool to monitor the fate of α-syn accumulation in the CNS and to evaluate the therapeutic effects of compounds targeting α-syn.

Neuroprotective effects of the anti-cancer drug sunitinib in models of HIV neurotoxicity suggests potential for the treatment of neurodegenerative disorders.

BACKGROUND AND PURPOSE: Anti-retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin-dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood-brain barrier. EXPERIMENTAL APPROACH: We screened a small group of known TKIs for a candidate with additional CDK5 inhibitory activity and tested the efficacy of the candidate in in vitro and in vivo models of HIV-gp120 neurotoxicity. KEY RESULTS: Among 12 different compounds, sunitinib inhibited CDK5 with an IC50 of 4.2 µM. In silico analysis revealed that, similarly to roscovitine, sunitinib fitted 6 of 10 features of the CDK5 pharmacophore. In a cell-based model, sunitinib reduced CDK5 phosphorylation (pCDK5), calpain-dependent p35/p25 conversion and protected neuronal cells from the toxic effects of gp120. In glial fibrillary acidic protein-gp120 transgenic (tg) mice, sunitinib reduced levels of pCDK5, p35/p25 and phosphorylated tau protein, along with amelioration of the neurodegenerative pathology. CONCLUSIONS AND IMPLICATIONS: Compounds such as sunitinib with dual kinase inhibitory activity could ameliorate the cognitive impairment associated with chronic HIV infection of the CNS. Moreover, repositioning existing low MW compounds holds promise for the treatment of patients with neurodegenerative disorders.

Hippocampal neuronal cells that accumulate α-synuclein fragments are more vulnerable to Aß oligomer toxicity via mGluR5--implications for dementia with Lewy bodies.

BACKGROUND: In dementia with Lewy bodies (DLB) abnormal interactions between α-synuclein (α-syn) and beta amyloid (Aß) result in selective degeneration of neurons in the neocortex, limbic system and striatum. However, factors rendering these neurons selectively vulnerable have not been fully investigated. The metabotropic glutamate receptor 5 (mGluR5) has been shown to be up regulated in DLB and might play a role as a mediator of the neurotoxic effects of Aß and α-syn in vulnerable neuronal populations. In this context, the main objective of the present study was to investigate the role of mGluR5 as a mediator of the neurotoxic effects of α-syn and Aß in the hippocampus. RESULTS: We generated double transgenic mice over-expressing amyloid precursor protein (APP) and α-syn under the mThy1 cassette and investigated the relationship between α-syn cleavage, Aß, mGluR5 and neurodegeneration in the hippocampus. We found that compared to the single tg mice, the α-syn/APP tg mice displayed greater accumulation of α-syn and mGluR5 in the CA3 region of the hippocampus compared to the CA1 and other regions. This was accompanied by loss of CA3 (but not CA1) neurons in the single and α-syn/APP tg mice and greater loss of MAP 2 and synaptophysin in the CA3 in the α-syn/APP tg. mGluR5 gene transfer using a lentiviral vector into the hippocampus CA1 region resulted in greater α-syn accumulation and neurodegeneration in the single and α-syn/APP tg mice. In contrast, silencing mGluR5 with a lenti-shRNA protected neurons in the CA3 region of tg mice. In vitro, greater toxicity was observed in primary hippocampal neuronal cultures treated with Aß oligomers and over-expressing α-syn; this effect was attenuated by down-regulating mGluR5 with an shRNA lentiviral vector. In α-syn-expressing neuronal cells lines, Aß oligomers promoted increased intracellular calcium levels, calpain activation and α-syn cleavage resulting in caspase-3-dependent cell death. Treatment with pharmacological mGluR5 inhibitors such as 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) attenuated the toxic effects of Aß in α-syn-expressing neuronal cells. CONCLUSIONS: Together, these results support the possibility that vulnerability of hippocampal neurons to α-syn and Aß might be mediated via mGluR5. Moreover, therapeutical interventions targeting mGluR5 might have a role in DLB.

AC-186, a selective nonsteroidal estrogen receptor ß agonist, shows gender specific neuroprotection in a Parkinson's disease rat model.

Drugs that selectively activate estrogen receptor ß (ERß) are potentially safer than the nonselective estrogens currently used in hormonal replacement treatments that activate both ERß and ERα. The selective ERß agonist AC-186 was evaluated in a rat model of Parkinson's disease induced through bilateral 6-hydroxydopamine lesions of the substantia nigra. In this model, AC-186 prevented motor, cognitive, and sensorimotor gating deficits and mitigated the loss of dopamine neurons in the substantia nigra, in males, but not in females. Furthermore, in male rats, 17ß-estradiol, which activates ERß and ERα with equal potency, did not show the same neuroprotective benefits as AC-186. Hence, in addition to a beneficial safety profile for use in both males and females, a selective ERß agonist has a differentiated pharmacological profile compared to 17ß-estradiol in males.

Pimavanserin, a 5-HT2A receptor inverse agonist, reverses psychosis-like behaviors in a rodent model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration in cognitive functioning. Overall, 25-50% of patients with AD also show symptoms of psychosis including hallucinations and delusions. As all available antipsychotic drugs have a 'black-box' warning for use in these patients because of increased mortality, no appropriate treatment for psychotic symptoms in AD currently exists. In the present study, we examined whether selective antagonism of 5-HT(2A) serotonin receptors has antipsychotic-like activity in an animal model of AD. Mice receiving an intracerebroventricular infusion of the amyloid ß(25-35) peptide fragment showed AD-like histopathology and a psychosis-related behavioral phenotype with enhanced responses to the psychostimulants 2,5-dimethoxy-4-iodoamphetamine hydrochloride and amphetamine as well as disrupted prepulse inhibition. Treatment with pimavanserin, a selective serotonin 5-HT(2A) receptor inverse agonist, prevented 2,5-dimethoxy-4-iodoamphetamine hydrochloride-induced head twitches, reversed the augmented locomotor response to amphetamine, and normalized prepulse inhibition in mice with amyloid pathology. These data suggest that an infusion of amyloid ß might induce alterations in serotonergic function that underlie a psychosis-like phenotype that can be normalized by treatment with a 5-HT(2A) inverse agonist. This in turn suggests that 5-HT(2A) inverse agonists, such as pimavanserin, might have therapeutic benefits in the treatment of psychosis in AD patients.

Differences in the locomotor-activating effects of indirect serotonin agonists in habituated and non-habituated rats.

The indirect serotonin (5-HT) agonist 3,4-methylenedioxymethamphetamine (MDMA) produces a distinct behavioral profile in rats consisting of locomotor hyperactivity, thigmotaxis, and decreased exploration. The indirect 5-HT agonist α-ethyltryptamine (AET) produces a similar behavioral profile. Using the Behavioral Pattern Monitor (BPM), the present investigation examined whether the effects of MDMA and AET are dependent on the novelty of the testing environment. These experiments were conducted in Sprague-Dawley rats housed on a reversed light cycle and tested during the dark phase of the light/dark cycle. We found that racemic MDMA (RS-MDMA; 3 mg/kg, SC) increased locomotor activity in rats tested in novel BPM chambers, but had no effect on locomotor activity in rats habituated to the BPM chambers immediately prior to testing. Likewise, AET (5 mg/kg, SC) increased locomotor activity in non-habituated animals but not in animals habituated to the test chambers. These results were unexpected because previous reports indicate that MDMA has robust locomotor-activating effects in habituated animals. To further examine the influence of habituation on MDMA-induced locomotor activity, we conducted parametric studies with S-(+)-MDMA (the more active enantiomer) in habituated and non-habituated rats housed on a standard or reversed light cycle. Light cycle was included as a variable due to reported differences in sensitivity to serotonergic ligands during the dark and light phases. In confirmation of our initial studies, rats tested during the dark phase and habituated to the BPM did not show an S-(+)-MDMA (3 mg/kg, SC)-induced increase in locomotor activity, whereas non-habituated rats did. By contrast, in rats tested during the light phase, S-(+)-MDMA increased locomotor activity in both non-habituated and habituated rats, although the response in habituated animals was attenuated. The finding that habituation and light cycle interact to influence MDMA- and AET-induced hyperactivity demonstrates that there are previously unrecognized complexities associated with the behavioral effects of these drugs.

Pimavanserin, a 5-HT2A inverse agonist, reverses psychosis-like behaviors in a rodent model of Parkinson's disease.

Parkinson's disease psychosis (PDP) is a condition for which a safe, tolerated, and effective therapy is lacking. Treatment with typical or atypical antipsychotics may be contraindicated in patients with PDP because of the potential for aggravating motor symptoms. This study used a novel animal model with features of both Parkinson's disease (PD) and psychosis to examine a potential mechanism for reversing PDP. Animals with bilateral 6-hydroxydopamine lesions of the substantia nigra displayed motoric impairments characteristic of humans with PD. In addition, they displayed augmented head twitches, augmented amphetamine-induced locomotor activity, and disrupted prepulse inhibition compared with sham controls, behavioral indices frequently used to assess antipsychotic activity in animal models. Pimavanserin, a selective 5-HT2A antagonist/inverse agonist, reversed the psychotic-like behavioral deficits, suggesting that nigrostriatal (6-hydroxydopamine) lesions induced alterations in 5-HT2A-mediated signaling. The selective 5-HT2A inverse agonist M100907, but not the selective 5-HT2C inverse agonist SB 252084 paralleled the effects of pimavanserin. Of note, the reversal of psychotic-like behaviors produced by 5-HT2A inverse agonists occurred without disrupting motor behaviors in lesioned subjects, suggesting that 5HT2A antagonism/inverse agonism may be beneficial in the treatment of PDP.

Alterations in mGluR5 expression and signaling in Lewy body disease and in transgenic models of alpha-synucleinopathy--implications for excitotoxicity.

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are neurodegenerative disorders of the aging population characterized by the abnormal accumulation of alpha-synuclein (alpha-syn). Previous studies have suggested that excitotoxicity may contribute to neurodegeneration in these disorders, however the underlying mechanisms and their relationship to alpha-syn remain unclear. For this study we proposed that accumulation of alpha-syn might result in alterations in metabotropic glutamate receptors (mGluR), particularly mGluR5 which has been linked to deficits in murine models of PD. In this context, levels of mGluR5 were analyzed in the brains of PD and DLB human cases and alpha-syn transgenic (tg) mice and compared to age-matched, unimpaired controls, we report a 40% increase in the levels of mGluR5 and beta-arrestin immunoreactivity in the frontal cortex, hippocampus and putamen in DLB cases and in the putamen in PD cases. In the hippocampus, mGluR5 was more abundant in the CA3 region and co-localized with alpha-syn aggregates. Similarly, in the hippocampus and basal ganglia of alpha-syn tg mice, levels of mGluR5 were increased and mGluR5 and alpha-syn were co-localized and co-immunoprecipitated, suggesting that alpha-syn interferes with mGluR5 trafficking. The increased levels of mGluR5 were accompanied by a concomitant increase in the activation of downstream signaling components including ERK, Elk-1 and CREB. Consistent with the increased accumulation of alpha-syn and alterations in mGluR5 in cognitive- and motor-associated brain regions, these mice displayed impaired performance in the water maze and pole test, these behavioral alterations were reversed with the mGluR5 antagonist, MPEP. Taken together the results from study suggest that mGluR5 may directly interact with alpha-syn resulting in its over activation and that this over activation may contribute to excitotoxic cell death in select neuronal regions. These results highlight the therapeutic importance of mGluR5 antagonists in alpha-synucleinopathies.

Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury.

Reactive astrocytes in neurotrauma, stroke, or neurodegeneration are thought to undergo cellular hypertrophy, based on their morphological appearance revealed by immunohistochemical detection of glial fibrillary acidic protein, vimentin, or nestin, all of them forming intermediate filaments, a part of the cytoskeleton. Here, we used a recently established dye-filling method to reveal the full three-dimensional shape of astrocytes assessing the morphology of reactive astrocytes in two neurotrauma models. Both in the denervated hippocampal region and the lesioned cerebral cortex, reactive astrocytes increased the thickness of their main cellular processes but did not extend to occupy a greater volume of tissue than nonreactive astrocytes. Despite this hypertrophy of glial fibrillary acidic protein-containing cellular processes, interdigitation between adjacent hippocampal astrocytes remained minimal. This work helps to redefine the century-old concept of hypertrophy of reactive astrocytes.

Collaborative development of the Arrowsmith two node search interface designed for laboratory investigators.

Arrowsmith is a unique computer-assisted strategy designed to assist investigators in detecting biologically-relevant connections between two disparate sets of articles in Medline. This paper describes how an inter-institutional consortium of neuroscientists used the UIC Arrowsmith web interface http://arrowsmith.psych.uic.edu in their daily work and guided the development, refinement and expansion of the system into a suite of tools intended for use by the wider scientific community.

High-resolution large-scale mosaic imaging using multiphoton microscopy to characterize transgenic mouse models of human neurological disorders.

The thorough characterization of transgenic mouse models of human central nervous system diseases is a necessary step in realizing the full benefit of using animal models to investigate disease processes and potential therapeutics. Because of the labor- and resource-intensive nature of high-resolution imaging, detailed investigation of possible structural or biochemical alterations in brain sections has typically focused on specific regions of interest as determined by the researcher a priori. For example, Parkinson's disease researchers often focus imaging on regions of the brain expected to exhibit pathology such as the substantia nigra and striatum. Because of limitations in acquiring and storing high-resolution imaging data, additional data contained in the specimen is not usually acquired or disseminated/reported to the research community. Here we present a method of imaging large regions of brain at close to the resolution limit of light microscopy using a mosaic imaging technique in conjunction with multiphoton microscopy. These maps are being used to characterize several genetically modified animal models of neurological disease by filling the information "gap" among techniques such as magnetic resonance imaging and electron microscopic analysis.

The cell-centered database: a database for multiscale structural and protein localization data from light and electron microscopy.

The creation of structured shared data repositories for molecular data in the form of web-accessible databases like GenBank has been a driving force behind the genomic revolution. These resources serve not only to organize and manage molecular data being created by researchers around the globe, but also provide the starting point for data mining operations to uncover interesting information present in the large amount of sequence and structural data. To realize the full impact of the genomic and proteomic efforts of the last decade, similar resources are needed for structural and biochemical complexity in biological systems beyond the molecular level, where proteins and macromolecular complexes are situated within their cellular and tissue environments. In this review, we discuss our efforts in the development of neuroinformatics resources for managing and mining cell level imaging data derived from light and electron microscopy. We describe the main features of our web-accessible database, the Cell Centered Database (CCDB; http://ncmir.ucsd.edu/CCDB/), designed for structural and protein localization information at scales ranging from large expanses of tissue to cellular microdomains with their associated macromolecular constituents. The CCDB was created to make 3D microscopic imaging data available to the scientific community and to serve as a resource for investigating structural and macromolecular complexity of cells and tissues, particularly in the rodent nervous system.

Distribution of rSlo Ca2+-activated K+ channels in rat astrocyte perivascular endfeet.

Evidence that Ca(2+)-activated K(+) (K(Ca)) channels play a role in cell volume changes and K(+) homeostasis led to a prediction that astrocytes would have K(Ca) channels near blood vessels in order to maintain K(+) homeostasis. Consistent with this thinking the present study demonstrates that rSlo K(Ca) channels are in glial cells of the adult rat central nervous system (CNS) and highly localized to specializations of astrocytes associated with the brain vasculature. Using confocal and thin-section electron microscopic immunolabeling methods the distribution of rSlo was examined in adult rat brain. Strong rSlo immunolabeling was present around the vasculature of most brain regions. Examination of dye-filled hippocampal astrocytes revealed rSlo immunolabeling polarized in astrocytic endfeet. Ultrastructural analysis confirmed that the rSlo staining was concentrated in astrocytic endfeet ensheathing capillaries as well as abutting the pia mater. Immunostaining within the endfeet was predominantly distributed at the plasma membrane directly adjacent to either the vascular basal lamina or the pial surface. The distribution of the aquaporin-4 (AQP-4) water channel was also examined using dye-filled hippocampal astrocytes. In confirmation of earlier reports, intense AQP-4 immunolabeling was generally observed at the perimeter of blood vessels, and coincided with perivascular endfeet and rSlo labeling. We propose that rSlo K(Ca) channels, with their sensitivity to membrane depolarization and intracellular calcium, play a role in the K(+) modulation of cerebral blood flow. Additional knowledge of the molecular and cellular machinery present at perivascular endfeet may provide insight into the structural and functional molecular elements responsible for the neuronal activity-dependent regulation of cerebral blood flow.