Considering Decoupled Phenotypic Diversification Between Ontogenetic Phases in Macroevolution: An Example Using Triggerfishes (Balistidae).

Across the Tree of Life, most studies of phenotypic disparity and diversification have been restricted to adult organisms. However, many lineages have distinct ontogenetic phases that differ from their adult forms in morphology and ecology. Focusing disproportionately on the evolution of adult forms unnecessarily hinders our understanding of the pressures shaping evolution over time. Non-adult disparity patterns are particularly important to consider for coastal ray-finned fishes, which can have juvenile phases with distinct phenotypes. These juvenile forms are often associated with sheltered nursery environments, with phenotypic shifts between adults and juvenile stages that are readily apparent in locomotor morphology. Whether this ontogenetic variation in locomotor morphology reflects a decoupling of diversification dynamics between life stages remains unknown. Here we investigate the evolutionary dynamics of locomotor morphology between adult and juvenile triggerfishes. We integrate a time-calibrated phylogenetic framework with geometric morphometric approaches and measurement data of fin aspect ratio and incidence, and reveal a mismatch between morphospace occupancy, the evolution of morphological disparity, and the tempo of trait evolution between life stages. Collectively, our results illuminate how the heterogeneity of morpho-functional adaptations can decouple the mode and tempo of morphological diversification between ontogenetic stages.

Differential behavioral outcomes following neonatal versus fetal human retinal pigment epithelial cell striatal implants in parkinsonian rats.

Following the failure of a Phase II clinical study evaluating human retinal pigment epithelial (hRPE) cell implants as a potential treatment option for Parkinson's disease, speculation has centered on implant function and survival as possible contributors to the therapeutic outcomes. We recently reported that neonatal hRPE cells, similar to hRPE cells used in the Phase II clinical study, produced short-lived in vitro and limited in vivo trophic factors, which supports that assumption. We hypothesize that the switch from fetal to neonatal hRPE cells, between the Phase I and the Phase II clinical trial may be partly responsible for the later negative outcomes. To investigate this hypothesis, we used two neonatal hRPE cell lots, prepared in a similar manner to neonatal hRPE cells used in the Phase II clinical study, and compared them to previously evaluated fetal hRPE cells for behavioral changes following unilateral striatal implantation in 6-hydroxydopamine-lesioned rats. The results showed that only fetal, not neonatal, hRPE cell implants, were able to improve behavioral outcomes following striatal implantation in the lesioned rats. These data suggest that fetal hRPE cells may be preferential to neonatal hRPE cells in restoring behavioral deficits.

Neonatal human retinal pigment epithelial cells secrete limited trophic factors in vitro and in vivo following striatal implantation in parkinsonian rats.

Human retinal pigment epithelial (hRPE) cell implants into the striatum have been investigated as a potential cell-based treatment for Parkinson's disease in a Phase II clinical trial that recently failed. We hypothesize that the trophic factor potential of the hRPE cells could potentially influence the function and/or survival of the implants and may be involved in an alternative mechanism of action. However, it is unclear if hRPE cells secreted trophic factors when handled in the manner used in the clinical Phase II trial. To address these questions, we investigated two neonatal hRPE cell lots, cultured in a similar manner to hRPE cells used in a Phase II clinical study, and longitudinally determined brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2), and pigment epithelium-derived factor concentrations in vitro and following striatal implantation into 6-hydroxydopamine-lesioned rats. The results demonstrate short-lived BDNF and FGF2 concentrations in vitro from hRPE cells grown alone or attached to gelatin microcarriers (GM)s as well as limited trophic factor concentration differences in vivo following striatal implantation of hRPE-GM in 6-hydroxydopamine lesioned rats compared to sham (GM-only). The data suggest that trophic factors from neonatal hRPE cell implants likely did not participate in an alternative mechanism of action, which adds supports to a hypothesis that additional factors may have been necessary for the survival and/or function of hRPE implants and potentially the success of the Phase II clinical trial.

Caspase-1 inhibition improves cognition without significantly altering amyloid and inflammation in aged Alzheimer disease mice.

Human genetic and animal model studies indicate that brain microglial inflammation is a primary driver of cognitive impairment in Alzheimer Disease (AD). Inflammasome-activated Caspase-1 (Casp1) is associated with both AD microglial inflammation and neuronal degeneration. In mice, Casp1 genetic ablation or VX-765 small molecule inhibition of Casp1 given at onset of cognitive deficits strongly supports the association between microglial inflammation and cognitive impairment. Here, VX-765 significantly improved episodic and spatial memory impairment eight months after the onset of cognitive impairment in aged AD mice with significant amyloid beta peptide (Aß) accumulation and microglial inflammation. Unexpectedly, while cognitive improvement was associated with dendritic spine density and hippocampal synaptophysin level recovery, VX-765 only slightly decreased Aß deposition and did not alter biochemically-measured Aß levels. Furthermore, increased hippocampal Iba1+-microglia, GFAP+-astrocytes, IL-1ß, and TNF-α levels were unaltered by VX-765. These results support the hypothesis that neuronal degeneration, not Aß or microglial inflammation, drives cognitive impairment in AD.

Therapeutic potential of Nlrp1 inflammasome, Caspase-1, or Caspase-6 against Alzheimer disease cognitive impairment.

The sequential activation of Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing protein 1 (Nlrp1) inflammasome, Caspase-1 (Casp1), and Caspase-6 (Casp6) is implicated in primary human neuron cultures and Alzheimer Disease (AD) neurodegeneration. To validate the Nlrp1-Casp1-Casp6 pathway in vivo, the APPSwedish/Indiana J20 AD transgenic mouse model was generated on either a Nlrp1, Casp1 or Casp6 null genetic background and mice were studied at 4-5 months of age. Episodic memory deficits assessed with novel object recognition were normalized by genetic ablation of Nlrp1, Casp1, or Casp6 in J20 mice. Spatial learning deficits, assessed with the Barnes Maze, were normalized in genetically ablated J20, whereas memory recall was normalized in J20/Casp1-/- and J20/Casp6-/-, and improved in J20/Nlrp1-/- mice. Hippocampal CA1 dendritic spine density of the mushroom subtype was reduced in J20, and normalized in genetically ablated J20 mice. Reduced J20 hippocampal dentate gyrus and CA3 synaptophysin levels were normalized in genetically ablated J20. Increased Iba1+-microglia in the hippocampus and cortex of J20 brains were normalized by Casp1 and Casp6 ablation and reduced by Nlrp1 ablation. Increased pro-inflammatory cytokines, TNF-α and CXCL1, in the J20 hippocampus were normalized by Nlrp1 or Casp1 genetic ablation. CXCL1 was also normalized by Casp6 genetic ablation. IFN-γ was increased and total amyloid ß peptide was decreased in genetically ablated Nlrp1, Casp1 or Casp6 J20 hippocampi. We conclude that Nlrp1, Casp1, or Casp6 are implicated in AD-related cognitive impairment, inflammation, and amyloidogenesis. These results indicate that Nlrp1, Casp1, and Casp6 represent rational therapeutic targets against cognitive impairment and inflammation in AD.

Pre-symptomatic Caspase-1 inhibitor delays cognitive decline in a mouse model of Alzheimer disease and aging.

Early therapeutic interventions are essential to prevent Alzheimer Disease (AD). The association of several inflammation-related genetic markers with AD and the early activation of pro-inflammatory pathways in AD suggest inflammation as a plausible therapeutic target. Inflammatory Caspase-1 has a significant impact on AD-like pathophysiology and Caspase-1 inhibitor, VX-765, reverses cognitive deficits in AD mouse models. Here, a one-month pre-symptomatic treatment of Swedish/Indiana mutant amyloid precursor protein (APPSw/Ind) J20 and wild-type mice with VX-765 delays both APPSw/Ind- and age-induced episodic and spatial memory deficits. VX-765 delays inflammation without considerably affecting soluble and aggregated amyloid beta peptide (Aß) levels. Episodic memory scores correlate negatively with microglial activation. These results suggest that Caspase-1-mediated inflammation occurs early in the disease and raise hope that VX-765, a previously Food and Drug Administration-approved drug for human CNS clinical trials, may be a useful drug to prevent the onset of cognitive deficits and brain inflammation in AD.

Sustainability of an in-home fire prevention intervention.

Residential fires remain a challenge in many parts of the United States. This project assessed the sustainability of a community-based fire prevention intervention on household fire safety knowledge and practices. The design was a prospective, cohort study including preintervention and postintervention surveys, which assessed participants' fire safety knowledge and behavior. The implementation of an in-home visit to educate parents of third- and fourth-grade students on escape planning coupled with the installation of smoke alarms can be successful in increasing basic fire safety knowledge and household fire safety practices.

Methylene blue inhibits Caspase-6 activity, and reverses Caspase-6-induced cognitive impairment and neuroinflammation in aged mice.

Activated Caspase-6 (Casp6) is associated with age-dependent cognitive impairment and Alzheimer disease (AD). Mice expressing human Caspase-6 in hippocampal CA1 neurons develop age-dependent cognitive deficits, neurodegeneration and neuroinflammation. This study assessed if methylene blue (MB), a phenothiazine that inhibits caspases, alters Caspase-6-induced neurodegeneration and cognitive impairment in mice. Aged cognitively impaired Casp6-overexpressing mice were treated with methylene blue in drinking water for 1 month. Methylene blue treatment did not alter Caspase-6 levels, assessed by RT-PCR, western blot and immunohistochemistry, but inhibited fluorescently-labelled Caspase-6 activity in acute brain slice intact neurons. Methylene blue treatment rescued Caspase-6-induced episodic and spatial memory deficits measured by novel object recognition and Barnes maze, respectively. Methylene blue improved synaptic function of hippocampal CA1 neurons since theta-burst long-term potentiation (LTP), measured by field excitatory postsynaptic potentials (fEPSPs) in acute brain slices, was successfully induced in the Schaffer collateral-CA1 pathway in methylene blue-treated, but not in vehicle-treated, Caspase-6 mice. Increased neuroinflammation, measured by ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia numbers and subtypes, and glial fibrillary acidic protein (GFAP)-positive astrocytes, were decreased by methylene blue treatment. Therefore, methylene blue reverses Caspase-6-induced cognitive deficits by inhibiting Caspase-6, and Caspase-6-mediated neurodegeneration and neuroinflammation. Our results indicate that Caspase-6-mediated damage is reversible months after the onset of cognitive deficits and suggest that methylene blue could benefit Alzheimer disease patients by reversing Caspase-6-mediated cognitive decline.

Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is an intractable progressive neurodegenerative disease characterized by cognitive decline and dementia. An inflammatory neurodegenerative pathway, involving Caspase-1 activation, is associated with human age-dependent cognitive impairment and several classical AD brain pathologies. Here, we show that the nontoxic and blood-brain barrier permeable small molecule Caspase-1 inhibitor VX-765 dose-dependently reverses episodic and spatial memory impairment, and hyperactivity in the J20 mouse model of AD. Cessation of VX-765 results in the reappearance of memory deficits in the mice after 1 month and recommencement of treatment re-establishes normal cognition. VX-765 prevents progressive amyloid beta peptide deposition, reverses brain inflammation, and normalizes synaptophysin protein levels in mouse hippocampus. Consistent with these findings, Caspase-1 null J20 mice are protected from episodic and spatial memory deficits, neuroinflammation and Aß accumulation. These results provide in vivo proof of concept for Caspase-1 inhibition against AD cognitive deficits and pathologies.

Human retinal pigment epithelial cell implants ameliorate motor deficits in two rat models of Parkinson disease.

Intrastriatal transplantation of gelatin microcarrier-attached human retinal pigment epithelial cells (hRPE-GM) may represent an alternative source for cell therapy in Parkinson disease (PD). The use of human retinal pigment epithelial (hRPE) cells in PD relies on the capacity of these cells to produce l-dopa as an intermediate product in the eumelanin synthesis pathway. We investigated the behavioral effects of hRPE-GM implants on forelimb use asymmetries and hindlimb motor deficits in unilateral and bilateral 6-hydroxydopamine (6-OHDA) rat models of PD. We report that intrastriatal unilateral implantation of hRPE-GM in rats with 6-OHDA nigrostriatal lesions produce an amelioration of the contralateral forelimb disuse and the contralateral hindlimb deficits. These results further support the possibility that implantation of cultured hRPE cells may be a promising therapeutic option for patients with PD.

Characterization and survival of long-term implants of human retinal pigment epithelial cells attached to gelatin microcarriers in a model of Parkinson disease.

Previous studies have demonstrated that the intrastriatal implantation of human retinal pigment epithelial cells attached to gelatin microcarriers (hRPE-GM) ameliorates behavioral deficits in animal models of Parkinson disease. However, there are only sparse data on cell survival in the host. In this study, we characterized a variety of retinal pigment epithelial (RPE)-specific markers in vitro and used these markers to investigate the long-term survival of hRPE-GM implants. Sprague-Dawley rats (n = 22) were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) and implanted with hRPE-GM without immunosuppression. Rats were euthanized at 48 hours, 7 days, 4 weeks, and 5 months postimplant and immunohistochemically processed using the following antibodies: 1) human-specific nuclear mitotic apparatus protein (NuMA-Ab2), 2) epithelial-specific extracellular matrix metalloproteinase inducer (EMMPRIN), 3) RPE cell-specific RPE65, and the inflammation markers 4) glial fibrillary acidic protein and 5) ED1 (rat CD68). Our analysis revealed NuMA-, EMMPRIN-, and RPE65-immunoreactive cells at different times postimplant. The morphologic features of hRPE cell implants (at 48 hours and 5 months) were confirmed by electron microscopy. Furthermore, despite evidence of chronic inflammation at the later time point, there is an appreciable number of surviving hRPE cells. This study suggests that hRPE-GM implants can survive in the absence of immunosuppression and can be potentially used as an alternative for treating Parkinson disease.

A comparison of blood gases, biochemistry, and hematology to ecomorphology in a health assessment of pinfish (Lagodon rhomboides).

Despite the promise of hematological parameters and blood chemistry in monitoring the health of marine fishes, baseline data is often lacking for small fishes that comprise central roles in marine food webs. This study establishes blood chemistry and hematological baseline parameters for the pinfish Lagodon rhomboides, a small marine teleost that is among the most dominant members of near-shore estuarine communities of the Atlantic Ocean and Gulf of Mexico. Given their prominence, pinfishes are an ideal candidate species to use as a model for monitoring changes across a wide range of near-shore marine communities. However, pinfishes exhibit substantial morphological differences associated with a preference for feeding in primarily sea-grass or sand dominated habitats, suggesting that differences in the foraging ecology of individuals could confound health assessments. Here we collect baseline data on the blood physiology of pinfish while assessing the relationship between blood parameters and measured aspects of feeding morphology using data collected from 37 individual fish. Our findings provide new baseline health data for this important near shore fish species and find no evidence for a strong linkage between blood physiology and either sex or measured aspects of feeding morphology. Comparing our hematological and biochemical data to published results from other marine teleost species suggests that analyses of trends in blood value variation correlated with major evolutionary transitions in ecology will shed new light on the physiological changes that underlie the successful diversification of fishes.

Modulation of Abnormal Metabolic Brain Networks by Experimental Therapies in a Nonhuman Primate Model of Parkinson Disease: An Application to Human Retinal Pigment Epithelial Cell Implantation.

Abnormal covariance pattern of regional metabolism associated with Parkinson disease (PD) is modulated by dopaminergic pharmacotherapy. Using high-resolution 18F-FDG PET and network analysis, we previously derived and validated a parkinsonism-related metabolic pattern (PRP) in nonhuman primate models of PD. It is currently not known whether this network is modulated by experimental therapeutics. In this study, we examined changes in network activity by striatal implantation of human levodopa-producing retinal pigment epithelial (hRPE) cells in parkinsonian macaques and evaluated the reproducibility of network activity in a small test-retest study. METHODS: 18F-FDG PET scans were acquired in 8 healthy macaques and 8 macaques with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced bilateral nigrostriatal dopaminergic lesions after unilateral putaminal implantation of hRPE cells or sham surgery. PRP activity was measured prospectively in all animals and in a subset of test-retest animals using a network quantification approach. Network activity and regional metabolic values were compared on a hemispheric basis between animal groups and treatment conditions. RESULTS: All individual macaques showed clinical improvement after hRPE cell implantation compared with the sham surgery. PRP activity was elevated in the untreated MPTP hemispheres relative to those of the normal controls (P < 0.00005) but was reduced (P < 0.05) in the hRPE-implanted hemispheres. The modulation observed in network activity was supported by concurrent local and remote changes in regional glucose metabolism. PRP activity remained unchanged in the untreated MPTP hemispheres versus the sham-operated hemispheres. PRP activity was also stable (P ≥ 0.29) and correlated (R2 ≥ 0.926; P < 0.00005) in the test-retest hemispheres. These findings were highly reproducible across several PRP topographies generated in multiple cohorts of parkinsonian and healthy macaques. CONCLUSION: We have demonstrated long-term therapeutic effects of hRPE cell implantation in nonhuman primate models of PD. The implantation of such levodopa-producing cells can concurrently decrease the elevated metabolic network activity in parkinsonian brains on an individual basis. These results parallel the analogous findings reported in patients with PD undergoing levodopa therapy and other symptomatic interventions. With further validation in large samples, 18F-FDG PET imaging with network analysis may provide a viable biomarker for assessing treatment response in animal models of PD after experimental therapies.

Blockade of stress-induced but not cocaine-induced reinstatement by infusion of noradrenergic antagonists into the bed nucleus of the stria terminalis or the central nucleus of the amygdala.

Experiments in our laboratory have shown that central noradrenergic (NA) activation plays a major role in stress-induced reinstatement of drug seeking in rats. In the present experiments, we investigated the effects of blockade of beta-NA adrenoceptors in the bed nucleus of the stria terminalis (BNST) and in the region of the central nucleus of the amygdala (CeA) on footshock- and cocaine-induced reinstatement. Rats were trained to self-administer cocaine (0.5 mg/kg, i.v.) for 9 d and, after a 5-7 d drug-free period, were given extinction sessions followed by a test for footshock stress-induced (15 min of intermittent footshock, 0.8 mA) or cocaine-induced (20 mg/kg, i.p.) reinstatement. Before the test, different groups of rats were given bilateral infusions of one of four doses of a mixture of the beta(1)- and beta(2)-receptor antagonists betaxolol and ICI-118,551 (vehicle, 0.25, 0.5, and 1 nmol of each compound in 0.5 microliter) into either the BNST or CeA. We observed a dose-dependent reduction of stress-induced reinstatement after infusions into the BNST and a complete blockade of stress-induced reinstatement after infusions into the CeA at all doses tested. The same treatments did not block cocaine-induced reinstatement when given at either site. These data suggest that stress-induced NA activation in the BNST and in the region of the CeA is critical to relapse to drug seeking induced by stress but not to relapse induced by priming injections of cocaine, and we hypothesize that NA activity leads to activation of corticotropin-releasing factor neurons in these regions.

Effects of cocaine in rats exposed to heroin.

We investigated whether chronic exposure to heroin alters responses to cocaine in ways that might explain the use of cocaine by opioid addicts. To this end, the effects of cocaine (5 and 20 mg/kg) were assessed on locomotor activity of rats chronically exposed to heroin (0.0, 3.5, 7.0, and 14.0 mg/kg/day, over 14 days, via osmotic mini-pumps), or withdrawn from heroin (1 day, acute withdrawal, and 14 days, protracted withdrawal). Chronic heroin exposure, in itself, dose dependently increased locomotion and acute cocaine administration further elevated locomotor activity in a dose-dependent and additive manner. During acute withdrawal, there was a dose-dependent decrease in locomotion that was reversed by cocaine in a dose-dependent manner. During protracted withdrawal, spontaneous locomotion normalized, but rats previously exposed to heroin displayed cross-sensitization to cocaine as indicated by small, but significant, enhanced locomotor response to 5 mg/kg of cocaine, and enhanced intravenous self-administration of low doses of cocaine (0.13 mg/kg/infusion). In a separate study, we measured extracellular dopamine (DA) in the nucleus accumbens (Acb) using in vivo microdialysis before and after acute withdrawal from heroin. During chronic exposure to heroin, basal extracellular DA was elevated dose dependently, whereas in acute withdrawal, levels were not different from those in vehicle-treated rats. In response to cocaine, however, DA activity in the Acb was significantly lower in rats withdrawn from the highest dose of heroin.

Direct intranigral administration of an ubiquitin proteasome system inhibitor in rat: behavior, positron emission tomography, immunohistochemistry.

Several independent lines of research suggest that disruption of the ubiquitin proteasome system (UPS) may play a role in the pathophysiology of Parkinson's disease. Direct intracerebral injection of UPS inhibitors (e.g. lactacystin) in animals has consistently produced important features of the disease. In this study, a range of lactacystin doses (0.5, 1, 2, 10 and 20 µg) were injected into the right substantia nigra in rats to determine the ideal dose required to produce a robust and specific lesion of the dopamine nigro-striatal system and motor deficits. Motor behavior, assessed with the tapered ledged beam task, was severely affected in animals that received high doses (10 and 20 µg) but only mild, impairments were observed in animals that received low doses (0.5, 1, and 2 µg). Positron emission tomography was performed with a dedicated small animal scanner on the rats following the injection of the radio-labeled tracer (±)[(11)C]dihydrotetrabenazine (DTBZ) which labels vesicular monoamine transporter type 2. Severe loss of [(11)C]DTBZ binding in the ipsilateral striatum was observed in the higher dose groups and mild loss was observed in the low dose groups. Stereological cell counting of tyrosine hydroxylase immunoreactive cells in the substantia nigra and the ventral tegmental area indicated a dose dependent loss of dopaminergic neurons. Significant correlations were found between the behavioral motor deficits, striatal [(11)C]DTBZ binding and cell counts of tyrosine hydroxylase immunoreactive cells. Taken together these results indicate that intranigral injection of lactacystin produces dose dependent effects on the dopamine nigro-striatal system and a dose of 10 µg will produce a consistent severe lesion.