Specific contribution of Reelin expressed by Cajal-Retzius cells or GABAergic interneurons to cortical lamination.

The extracellular protein Reelin, expressed by Cajal-Retzius (CR) cells at early stages of cortical development and at late stages by GABAergic interneurons, regulates radial migration and the "inside-out" pattern of positioning. Current models of Reelin functions in corticogenesis focus on early CR cell-derived Reelin in layer I. However, developmental disorders linked to Reelin deficits, such as schizophrenia and autism, are related to GABAergic interneuron-derived Reelin, although its role in migration has not been established. Here we selectively inactivated the Reln gene in CR cells or GABAergic interneurons. We show that CR cells have a major role in the inside-out order of migration, while CR and GABAergic cells sequentially cooperate to prevent invasion of cortical neurons into layer I. Furthermore, GABAergic cell-derived Reelin compensates some features of the reeler phenotype and is needed for the fine tuning of the layer-specific distribution of cortical neurons. In the hippocampus, the inactivation of Reelin in CR cells causes dramatic alterations in the dentate gyrus and mild defects in the hippocampus proper. These findings lead to a model in which both CR and GABAergic cell-derived Reelin cooperate to build the inside-out order of corticogenesis, which might provide a better understanding of the mechanisms involved in the pathogenesis of neuropsychiatric disorders linked to abnormal migration and Reelin deficits.

The armadillo-repeat containing X-linked protein 3, ARMCX3, is a negative regulator of the browning of adipose tissue associated with obesity.

OBJECTIVES: To determine the role of armadillo repeat-containing X-linked protein 3 (ARMCX3) in the thermogenic plasticity of adipose tissue. METHODS: Adipose tissues were characterized in Armcx3-KO male mice. Armcx3 gene expression was analyzed in adipose tissue from mice exposed to thermogenic inducers (cold, ß3-adenergic stimulus) and in differentiating brown and beige cells in culture. Analyses encompassed circulating metabolite and hormonal profiling, tissue characterization, histology, gene expression patterns, and immunoblot assays. Armcx3 gene expression was assessed in subcutaneous adipose tissue from lean individuals and individuals with obesity and was correlated with expression of marker genes of adipose browning. The effects of adenoviral-mediated overexpression of ARMCX3 on differentiating brown adipocyte gene expression and respiratory activity were determined. RESULTS: Male mice lacking ARMCX3 showed significant induction of white adipose tissue browning. In humans, ARMCX3 expression in subcutaneous adipose tissue was inversely correlated with the expression of marker genes of thermogenic activity, including CIDEA, mitochondrial transcripts, and creatine kinase-B. Armcx3 expression in adipose tissues was repressed by thermogenic activation (cold or ß3-adrenergic stimulation) and was upregulated by obesity in mice and humans. Experimentally-induced increases in Armcx3 caused down-regulation of thermogenesis-related genes and reduced mitochondrial oxidative activity of adipocytes in culture, whereas siRNA-mediated Armcx3 knocking-down enhanced expression of thermogenesis-related genes. CONCLUSION: ARMCX3 is a novel player in the control of thermogenic adipose tissue plasticity that acts to repress acquisition of the browning phenotype and shows a direct association with indicators of obesity in mice and humans.

Minocycline reduces microgliosis and improves subcortical white matter function in a model of cerebral vascular disease.

Chronic cerebral hypoperfusion is a key mechanism associated with white matter disruption in cerebral vascular disease and dementia. In a mouse model relevant to studying cerebral vascular disease, we have previously shown that cerebral hypoperfusion disrupts axon-glial integrity and the distribution of key paranodal and internodal proteins in subcortical myelinated axons. This disruption of myelinated axons is accompanied by increased microglia and cognitive decline. The aim of the present study was to investigate whether hypoperfusion impairs the functional integrity of white matter, its relation with axon-glial integrity and microglial number, and whether by targeting microglia these effects can be improved. We show that in response to increasing durations of hypoperfusion, the conduction velocity of myelinated fibres in the corpus callosum is progressively reduced and that paranodal and internodal axon-glial integrity is disrupted. The number of microglial cells increases in response to hypoperfusion and correlates with disrupted paranodal and internodal integrity and reduced conduction velocities. Further minocycline, a proposed anti-inflammatory and microglia inhibitor, restores white matter function related to a reduction in the number of microglia. The study suggests that microglial activation contributes to the structural and functional alterations of myelinated axons induced by cerebral hypoperfusion and that dampening microglia numbers/proliferation should be further investigated as potential therapeutic benefit in cerebral vascular disease.

Influence of Transgenic Metallothionein-1 on Gliosis, CA1 Neuronal Loss, and Brain Metal Levels of the Tg2576 Mouse Model of Alzheimer's Disease.

The mouse model of Alzheimer's disease (AD), Tg2576 mice (APP), has provided valuable information, such as the role of the metallothionein (MT) family in their behavioral and amyloidosis phenotypes. In this study, we further characterize the role of MT-1 by crossing Mt1-overexpressing mice with Tg2576 mice (APPTgMT). In 14-month-old mice, MT-1(/2) protein levels were dramatically increased by Mt1 overexpression throughout the cortex (Cx), which showed a prominent caudal-rostral gradient, and the hippocampus (HC). There was a trend for MT-1(/2) immunostaining to be increased in the areas surrounding the amyloid plaques in control male mice but not in Mt1-overexpressing mice. Gliosis was elicited by the amyloid plaques, but the effects of Mt1 overexpression were modest. However, in hippocampal western blots the microglial marker Iba-1 was increased in old male APPTgMT mice compared to APP-wild type (APPWT) mice, and the opposite was observed in young mice. Hippocampal CA1 neuronal loss was observed in Tg2576 mice, but was unaffected by Mt1 overexpression. Aging increased Zn and Cu levels differently depending on brain area, sex, and genotype. Thus, the effects of Mt1 overexpression on the phenotype of Tg2576 mice here studied are modest.

A mammalian-specific Alex3/Gαq protein complex regulates mitochondrial trafficking, dendritic complexity, and neuronal survival.

Mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neural activity. We investigated how G protein-coupled receptors (GPCRs) and G proteins control mitochondrial dynamics and trafficking. The activation of Gαq inhibited mitochondrial trafficking in neurons through a mechanism that was independent of the canonical downstream PLCß pathway. Mitoproteome analysis revealed that Gαq interacted with the Eutherian-specific mitochondrial protein armadillo repeat-containing X-linked protein 3 (Alex3) and the Miro1/Trak2 complex, which acts as an adaptor for motor proteins involved in mitochondrial trafficking along dendrites and axons. By generating a CNS-specific Alex3 knockout mouse line, we demonstrated that Alex3 was required for the effects of Gαq on mitochondrial trafficking and dendritic growth in neurons. Alex3-deficient mice had altered amounts of ER stress response proteins, increased neuronal death, motor neuron loss, and severe motor deficits. These data revealed a mammalian-specific Alex3/Gαq mitochondrial complex, which enables control of mitochondrial trafficking and neuronal death by GPCRs.

Characterization of the role of the antioxidant proteins metallothioneins 1 and 2 in an animal model of Alzheimer's disease.

Alzheimer's disease (AD) is by far the most commonly diagnosed dementia, and despite multiple efforts, there are still no effective drugs available for its treatment. One strategy that deserves to be pursued is to alter the expression and/or physiological action of endogenous proteins instead of administering exogenous factors. In this study, we intend to characterize the roles of the antioxidant, anti-inflammatory, and heavy-metal binding proteins, metallothionein-1 + 2 (MT1 + 2), in a mouse model of Alzheimer's disease, Tg2576 mice. Contrary to expectations, MT1 + 2-deficiency rescued partially the human amyloid precursor protein-induced changes in mortality and body weight in a gender-dependent manner. On the other hand, amyloid plaque burden was decreased in the cortex and hippocampus in both sexes, while the amyloid cascade, neuroinflammation, and behavior were affected in the absence of MT1 + 2 in a complex manner. These results highlight that the control of the endogenous production and/or action of MT1 + 2 could represent a powerful therapeutic target in AD.

Characterization of the role of metallothionein-3 in an animal model of Alzheimer's disease.

Among the dementias, Alzheimer's disease (AD) is the most commonly diagnosed, but there are still no effective drugs available for its treatment. It has been suggested that metallothionein-3 (MT-3) could be somehow involved in the etiology of AD, and in fact very promising results have been found in in vitro studies, but the role of MT-3 in vivo needs further analysis. In this study, we analyzed the role of MT-3 in a mouse model of AD, Tg2576 mice, which overexpress human Amyloid Precursor Protein (hAPP) with the Swedish mutation. MT-3 deficiency partially rescued the APP-induced mortality of females, and mildly affected APP-induced changes in behavior assessed in the hole-board and plus-maze tests in a gender-dependent manner. Amyloid plaque burden and/or hAPP expression were decreased in the cortex and hippocampus of MT-3-deficient females. Interestingly, exogenously administered Zn(7)MT-3 increased soluble Aß40 and Aß42 and amyloid plaques and gliosis, particularly in the cortex, and changed several behavioral traits (increased deambulation and exploration and decreased anxiety). These results highlight that the control of the endogenous production and/or action of MT-3 could represent a powerful therapeutic target in AD.

Adult-specific Reelin expression alters striatal neuronal organization: implications for neuropsychiatric disorders.

In addition to neuronal migration, brain development, and adult plasticity, the extracellular matrix protein Reelin has been extensively implicated in human psychiatric disorders such as schizophrenia, bipolar disorder, and autism spectrum disorder. Moreover, heterozygous reeler mice exhibit features reminiscent of these disorders, while overexpression of Reelin protects against its manifestation. However, how Reelin influences the structure and circuits of the striatal complex, a key region for the above-mentioned disorders, is far from being understood, especially when altered Reelin expression levels are found at adult stages. In the present study, we took advantage of complementary conditional gain- and loss-of-function mouse models to investigate how Reelin levels may modify adult brain striatal structure and neuronal composition. Using immunohistochemical techniques, we determined that Reelin does not seem to influence the striatal patch and matrix organization (studied by µ-opioid receptor immunohistochemistry) nor the density of medium spiny neurons (MSNs, studied with DARPP-32). We show that overexpression of Reelin leads to increased numbers of striatal parvalbumin- and cholinergic-interneurons, and to a slight increase in tyrosine hydroxylase-positive projections. We conclude that increased Reelin levels might modulate the numbers of striatal interneurons and the density of the nigrostriatal dopaminergic projections, suggesting that these changes may be involved in the protection of Reelin against neuropsychiatric disorders.

Metallothionein and brain inflammation.

Since the seminal discoveries of Bert Vallee regarding zinc and metallothioneins (MTs) more than 50 years ago, thousands of studies have been published concerning this fascinating story. One of the most active areas of research is the involvement of these proteins in the inflammatory response in general, and in neuroinflammation in particular. We describe the general aspects of the inflammatory response, highlighting the essential role of the major cytokine interleukin-6, and review briefly the expression and function of MTs in the central nervous system in the context of neuroinflammation. Particular attention is paid to the Tg2576 Alzheimer disease mouse model and the preliminary results obtained in mice into which human Zn(7)MT-2A was injected, which suggest a reversal of the behavioral deficits while enhancing amyloid plaque load and gliosis.

ARMCX3 Mediates Susceptibility to Hepatic Tumorigenesis Promoted by Dietary Lipotoxicity.

ARMCX3 is encoded by a member of the Armcx gene family and is known to be involved in nervous system development and function. We found that ARMCX3 is markedly upregulated in mouse liver in response to high lipid availability, and that hepatic ARMCX3 is upregulated in patients with NAFLD and hepatocellular carcinoma (HCC). Mice were subjected to ARMCX3 invalidation (inducible ARMCX3 knockout) and then exposed to a high-fat diet and diethylnitrosamine-induced hepatocarcinogenesis. The effects of experimental ARMCX3 knockdown or overexpression in HCC cell lines were also analyzed. ARMCX3 invalidation protected mice against high-fat-diet-induced NAFLD and chemically induced hepatocarcinogenesis. ARMCX3 invalidation promoted apoptotic cell death and macrophage infiltration in livers of diethylnitrosamine-treated mice maintained on a high-fat diet. ARMCX3 downregulation reduced the viability, clonality and migration of HCC cell lines, whereas ARMCX3 overexpression caused the reciprocal effects. SOX9 was found to mediate the effects of ARMCX3 in hepatic cells, with the SOX9 interaction required for the effects of ARMCX3 on hepatic cell proliferation. In conclusion, ARMCX3 is identified as a novel molecular actor in liver physiopathology and carcinogenesis. ARMCX3 downregulation appears to protect against hepatocarcinogenesis, especially under conditions of high dietary lipid-mediated hepatic insult.

The comparison of mouse full metallothionein-1 versus alpha and beta domains and metallothionein-1-to-3 mutation following traumatic brain injury reveals different biological motifs.

Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

Monoamine oxidase-B activity is not involved in the neuroinflammatory response elicited by a focal freeze brain injury.

Cryolesion of the frontoparietal cortex in mice is a well-described brain injury paradigm that results in increased astrogliosis surrounding the lesion site and is accompanied by a prominent increase in the MAO-B levels in astrocytes. Whether these increased MAO-B levels contribute to cellular damage or modulate reactive astrocytosis remains unclear. MAO-B activity may contribute to cellular damage, since its metabolism products are highly toxic to the cells. Additionally, it has been suggested that MAO-B inhibition may regulate astrocytic reaction. In this study, we have determined the relative contribution of MAO-B activity to the outcome following freeze injury. Freeze injury induced a prominent increase of several inflammatory markers, including ICAM, Mac-1, EB22, and GFAP. Inhibition of MAO-B activity using the selective inhibitor PF9601N did not reduce this cryolesion-induced inflammatory response. Additional data revealed that the expression of several cryolesion-induced cell death genes, such as Fas, Rip, p53, and ICE, was not reduced in PF9601N-treated mice, evidencing that MAO-B activity did not contribute to cryolesion-induced cell death. Definitive functional analysis of the mice using the ladder beam task revealed that MAO-B inhibition did not improve the cryolesion-induced motor impairment. These data strongly suggest that, although MAO-B is highly expressed in the area surrounding the lesion site, its activity does not contribute to the cellular damage or play any role in regulating astrocytic reactivity.

NEK7 regulates dendrite morphogenesis in neurons via Eg5-dependent microtubule stabilization.

Organization of microtubules into ordered arrays is best understood in mitotic systems, but remains poorly characterized in postmitotic cells such as neurons. By analyzing the cycling cell microtubule cytoskeleton proteome through expression profiling and targeted RNAi screening for candidates with roles in neurons, we have identified the mitotic kinase NEK7. We show that NEK7 regulates dendrite morphogenesis in vitro and in vivo. NEK7 kinase activity is required for dendrite growth and branching, as well as spine formation and morphology. NEK7 regulates these processes in part through phosphorylation of the kinesin Eg5/KIF11, promoting its accumulation on microtubules in distal dendrites. Here, Eg5 limits retrograde microtubule polymerization, which is inhibitory to dendrite growth and branching. Eg5 exerts this effect through microtubule stabilization, independent of its motor activity. This work establishes NEK7 as a general regulator of the microtubule cytoskeleton, controlling essential processes in both mitotic cells and postmitotic neurons.

Dimethyl fumarate improves white matter function following severe hypoperfusion: Involvement of microglia/macrophages and inflammatory mediators.

The brain's white matter is highly vulnerable to reductions in cerebral blood flow via mechanisms that may involve elevated microgliosis and pro-inflammatory pathways. In the present study, the effects of severe cerebral hypoperfusion were investigated on white matter function and inflammation. Male C57Bl/6J mice underwent bilateral common carotid artery stenosis and white matter function was assessed at seven days with electrophysiology in response to evoked compound action potentials (CAPs) in the corpus callosum. The peak latency of CAPs and axonal refractoriness was increased following hypoperfusion, indicating a marked functional impairment in white matter, which was paralleled by axonal and myelin pathology and increased density and numbers of microglia/macrophages. The functional impairment in peak latency was significantly correlated with increased microglia/macrophages. Dimethyl fumarate (DMF; 100 mg/kg), a drug with anti-inflammatory properties, was found to reduce peak latency but not axonal refractoriness. DMF had no effect on hypoperfusion-induced axonal and myelin pathology. The density of microglia/macrophages was significantly increased in vehicle-treated hypoperfused mice, whereas DMF-treated hypoperfused mice had similar levels to that of sham-treated mice. The study suggests that increased microglia/macrophages following cerebral hypoperfusion contributes to the functional impairment in white matter that may be amenable to modulation by DMF.

Long-term cilostazol treatment reduces gliovascular damage and memory impairment in a mouse model of chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion is a major cause of age-related vascular cognitive impairment. A well-characterised mouse model has shown that hypoperfusion results in gliovascular and white matter damage and impaired spatial working memory. In this study, we assessed whether cilostazol, a phosphodiesterase III inhibitor, could protect against these changes. Adult, male C57Bl/6J mice were subjected to bilateral common carotid artery stenosis or a sham operation and fed normal or cilostazol diet for three months. Cilostazol treatment reduced the impairment in working memory and white matter function after hypoperfusion. Endothelial adhesion molecules and gliosis, increased after hypoperfusion, were ameliorated with cilostazol treatment. Interestingly, the improvement in working memory was closely correlated with reduced microglia and endothelial adhesion molecules. Further, the number of stroke lesions after hypoperfusion was reduced in the cilostazol-treated group. Altogether cilostazol showed potential to ameliorate the gliovascular damage and working memory impairments after hypoperfusion possibly via endothelial protection supporting its potential use in the treatment of vascular cognitive impairment.

Overexpression of Metallothionein-1 Modulates the Phenotype of the Tg2576 Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most commonly diagnosed dementia, where signs of neuroinflammation and oxidative stress are prominent. In this study we intend to further characterize the roles of the antioxidant, anti-inflammatory, and heavy metal binding protein, metallothionein-1 (MT-1), by crossing Mt1 overexpressing mice with a well-known mouse model of AD, Tg2576 mice, which express the human amyloid-ß protein precursor (hAßPP) with the Swedish K670N/M671L mutations. Mt1 overexpression increased overall perinatal survival, but did not affect significantly hAßPP-induced mortality and weight loss in adult mice. Amyloid plaque burden in ∼14-month-old mice was increased by Mt1 overexpression in the hippocampus but not the cortex. Despite full length hAßPP levels and amyloid plaques being increased by Mt1 overexpression in the hippocampus of both sexes, oligomeric and monomeric forms of Aß, which may contribute more to toxicity, were decreased in the hippocampus of females and increased in males. Several behavioral traits such as exploration, anxiety, and learning were altered in Tg2576 mice to various degrees depending on the age and the sex. Mt1 overexpression ameliorated the effects of hAßPP on exploration in young females, and potentiated those on anxiety in old males, and seemed to improve the rate of spatial learning (Morris water maze) and the learning elicited by a classical conditioning procedure (eye-blink test). These results clearly suggest that MT-1 may be involved in AD pathogenesis.

Metallothionein and stress combine to affect multiple organ systems.

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that have a wide range of functions in cellular homeostasis and immunity. MTs can be induced by a variety of conditions including metals, glucocorticoids, endotoxin, acute phase cytokines, stress, and irradiation. In addition to their important immunomodulatory functions, MTs can protect essential cellular compartments from toxicants, serve as a reservoir of essential heavy metals, and regulate cellular redox potential. Many of the roles of MTs in the neuroinflammation, intestinal inflammation, and stress response have been investigated and were the subject of a session at the 6th International Congress on Stress Proteins in Biology and Medicine in Sheffield, UK. Like the rest of the cell stress response, there are therapeutic opportunities that arise from an understanding of MTs, and these proteins also provide potential insights into the world of the heat shock protein.

Restoration of oligodendrocyte pools in a mouse model of chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion, a sustained modest reduction in cerebral blood flow, is associated with damage to myelinated axons and cognitive decline with ageing. Oligodendrocytes (the myelin producing cells) and their precursor cells (OPCs) may be vulnerable to the effects of hypoperfusion and in some forms of injury OPCs have the potential to respond and repair damage by increased proliferation and differentiation. Using a mouse model of cerebral hypoperfusion we have characterised the acute and long term responses of oligodendrocytes and OPCs to hypoperfusion in the corpus callosum. Following 3 days of hypoperfusion, numbers of OPCs and mature oligodendrocytes were significantly decreased compared to controls. However following 1 month of hypoperfusion, the OPC pool was restored and increased numbers of oligodendrocytes were observed. Assessment of proliferation using PCNA showed no significant differences between groups at either time point but showed reduced numbers of proliferating oligodendroglia at 3 days consistent with the loss of OPCs. Cumulative BrdU labelling experiments revealed higher numbers of proliferating cells in hypoperfused animals compared to controls and showed a proportion of these newly generated cells had differentiated into oligodendrocytes in a subset of animals. Expression of GPR17, a receptor important for the regulation of OPC differentiation following injury, was decreased following short term hypoperfusion. Despite changes to oligodendrocyte numbers there were no changes to the myelin sheath as revealed by ultrastructural assessment and fluoromyelin however axon-glial integrity was disrupted after both 3 days and 1 month hypoperfusion. Taken together, our results demonstrate the initial vulnerability of oligodendroglial pools to modest reductions in blood flow and highlight the regenerative capacity of these cells.

Copper modulation as a therapy for Alzheimer's disease?

The role of metals in the pathophysiology of Alzheimer's disease (AD) has gained considerable support in recent years, with both in vitro and in vivo data demonstrating that a mis-metabolism of metal ions, such as copper and zinc, may affect various cellular cascades that ultimately leads to the development and/or potentiation of AD. In this paper, we will provide an overview of the preclinical and clinical literature that specifically relates to attempts to affect the AD cascade by the modulation of brain copper levels. We will also detail our own novel animal data, where we treated APP/PS1 (7-8 months old) mice with either high copper (20 ppm in the drinking water), high cholesterol (2% supplement in the food) or a combination of both and then assessed ß-amyloid (Aß) burden (soluble and insoluble Aß), APP levels and behavioural performance in the Morris water maze. These data support an interaction between copper/cholesterol and both Aß and APP and further highlight the potential role of metal ion dyshomeostasis in AD.