Marco Pugliese

ATP-dependent potassium channel blockade strengthens microglial neuroprotection after hypoxia–ischemia in rats

Stroke causes CNS injury associated with strong fast microglial activation as part of the inflammatory response. In rat models of stroke, sulphonylurea receptor blockade with glibenclamide reduced cerebral edema and infarct volume. We postulated that glibenclamide administered during the early stages of stroke might foster neuroprotective microglial activity through ATP-sensitive potassium (K(ATP)) channel blockade. We found in vitro that BV2 cell line showed upregulated expression of K(ATP) channel subunits in response to pro-inflammatory signals and that glibenclamide increases the reactive morphology of microglia, phagocytic capacity and TNFα release. Moreover, glibenclamide administered to rats 6, 12 and 24h after transient Middle Cerebral Artery occlusion improved neurological outcome and preserved neurons in the lesioned core three days after reperfusion. Immunohistochemistry with specific markers to neuron, astroglia, microglia and lymphocytes showed that resident amoeboid microglia are the main cell population in that necrotic zone. These reactive microglial cells express SUR1, SUR2B and Kir6.2 proteins that assemble in functional K(ATP) channels. These findings provide that evidence for the key role of K(ATP) channels in the control of microglial reactivity are consistent with a microglial effect of glibenclamide into the ischemic brain and suggest a neuroprotective role of microglia in the early stages of stroke.

γ-Aminobutyric acidergic interneuron vulnerability to aging in canine prefrontal cortex

The aged dog is considered a promising model for examining molecular and cellular processes involved in a variety of human neurological disorders. By using the canine counterpart of senile dementia of the Alzheimers type (ccSDAT), we investigated the specific vulnerability of the γ‐aminobutyric acid (GABA) cortical subset of interneurons, characterized by their calcium‐binding protein content, to neuronal death. Dogs representing a large variety of breeds were classified into three groups: young control, aged control, and ccSDAT. In all dogs, the general distribution and cell typology of parvalbumin‐, calretinin‐, and calbindin‐positive neurons were found to be similar to those in the human. As in Alzheimers disease patients, neurons displaying parvalbumin or calretinin immunoreactivity were resistant and the calbindin‐positive ones depleted. Together with aging, amyloid deposition in its early phase (stage II) participates in this specific neuronal death, but with a lower potency. In conclusion, our data provide evidence that preservation of GABAergic cortical interneurons has to be focused on the early stage of β‐amyloid deposition. We also demonstrate the usefulness of dogs of all breeds for investigating the early phases of human brain aging and Alzheimers disease.

Calcium precipitation in acute and chronic brain diseases.

In rat brain, calcification associated with excitotoxicity has been proposed to play a protective role, whereas in human brain, nonartherosclerotic calcification is present in several pathological conditions without any clear significance. To determine if calcification can be viewed as a protective step of calcium homeostasis during chronic and acute neuronal suffering, cerebral cortex and hippocampus of patients with Alzheimers disease, vascular dementia and neonatal hypoxia-ischemia were investigated. To investigate the human specificity, these two areas were also studied in dogs with established cognitive deficits. In all groups, calcium precipitates were observed in the cerebral parenchyma associated with neuronal damage. The cerebral cortex presented a higher degree of calcification than the hippocampus. The neonatal hypoxia-ischemia group was characterised by a higher degree of calcification, whereas the groups with lowest calcification were the Alzheimers patients and dogs. As shown by X-ray microanalysis, in the precipitates, calcium is mainly associated with phosphorus in a form that resembles hydroxyapatites. Thus, intracellular calcium concentration associated with neuronal suffering may reduce the energy extrusion. We propose that, to help overcome excitotoxicity, calcium precipitation acts in CNS of vertebrates as a new compartment of the calcium homeostasis in which free cytoplasmic calcium ions are inactivated by phosphate ones.

Diffuse beta-amyloid plaques and hyperphosphorylated tau are unrelated processes in aged dogs with behavioral deficits.

Single and double-labeling immunocytochemistry has been used to learn about the localization, distribution, and possible relationship between beta-amyloid protein (Aβ) deposition and tau hyperphosphorylation in the canine cerebral cortex with age. Behavioral impairment, as reported by the owners and tested in all dogs, correlated with increased Aβ burden in old dogs. Aβ plaques were diffuse and they were not accompanied by modifications in synaptic protein expression. Plaques were not associated with increased active mitogen activated protein kinase (MAPK/ERK-P) and p38 kinase (p38-P) expression, and tau hyperphosphorylation in neighboring cell processes. Yet tau hyperphosphorylation, as revealed with phospho-specific antibodies to tauThr181 and tauSer396, increased with age in individual neurons. Moreover, the subcellular pattern shifted from perinuclear localization to granular cytoplasmic and nuclear distribution with age. Our results in dog suggest that Aβ diffuse plaque formation and tau hyperphosphorylation are independent events, both occurring during the process of aging. Although increased cognitive dysfunction is associated with increased tau hyperphosphorylation, further investigation is needed to understand whether tau hyperphosphorylation is causative of cognitive impairment or an independent process related to aging.

Canine cognitive deficit correlates with diffuse plaque maturation and S100β (−) astrocytosis but not with insulin cerebrospinal fluid level

Like humans, canines develop with aging β-amyloid (Aβ) plaques and a progressive cognitive deficit on tasks similar to those used in diagnosis and follow-up of Alzheimer’s disease. Owing to that, dogs are quite unique to investigate the early events taking place in the diffuse Aβ plaque maturation and its relationship with cognitive deficit. The aim of the present investigation was to study the link between the diffuse Aβ plaque maturation and the astro- and microglial reactivity. The involvement of insulin and beta-subunit of S100 protein (S100β) overexpression in the process was also investigated. Aβ plaques were measured and counted in prefrontal cortex of 16 pet dogs of different breeds, weight and sex, classified as control and with a light or severe cognitive deficit. A correlation between canine graded cognitive deficit, diffuse plaque maturation, and S100β (−) astrocytosis, but not with cerebrospinal fluid insulin level, was found that may reflect the very early events of Aβ deposition in Alzheimer’s disease.

Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

BackgroundMultiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.MethodsAnti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNγ)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration.ResultsDiazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord.ConclusionTaken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.

Canine cognitive dysfunction and the cerebellum: Acetylcholinesterase reduction, neuronal and glial changes

The specific functional and pathological alterations observed in Alzheimers disease are less severe in the cerebellum than in other brain areas, particularly the entorhinal cortex and hippocampus. Since dense core amyloid-beta plaque formation has been associated with an acetylcholinesterase heterogeneous nucleator action, we examined if an acetylcholinesterase imbalance was involved in cerebellum plaque deposition. By using the canine counterpart of senile dementia of the Alzheimers type, a promising model of human brain aging and early phases of Alzheimers disease, we investigated how cerebellar pathology and acetylcholinesterase density could be related with cognitive dysfunction. As in Alzheimers disease, the late affectation of the cerebellum was evidenced by its lack of amyloid-beta plaque and the presence of diffuse deposition throughout all cortical grey matter layers. The highest acetylcholinesterase optic density corresponded to cerebellar islands of the granular layer and was predominantly associated with synaptic glomeruli and the somata of Golgi cells. Its reduction correlated with aging and loss of granule cells, whereas cognitive deficit only correlated with loss of Purkinje cells. The observed Bergmann glia alterations may correspond to a reactive response to the loss and damage of the Purkinje cells, their specific neuronal partner. Regarding the role of acetylcholinesterase mediation in amyloid-beta deposition, our data argue against an interaction between these two proteins because acetylcholinesterase reduction correlates with aging but not with cognitive deficit. Finally, our data support the use of companion dogs of all breeds to study aging and early phases of Alzheimers disease.

Dogs with Cognitive Dysfunction Syndrome: A Natural Model of Alzheimer’s Disease

In the search for appropriate models for Alzheimers disease (AD) involving animals other than rodents, several laboratories are working with animals that naturally develop cognitive dysfunction. Among the animals tested, dogs are quite unique in helping to elucidate the cascade of events that take place in brain amyloid-beta (Aβ)deposition aging, and cognitive deficit. Recent innovative research has validated human methods and tools for the analysis of canine neuropathology and has allowed the development of two different approaches to investigate dogs as natural models of AD. The first approach relates AD-like neuropathy with the decline in memory and learning ability in aged housed dogs in a highly controlled laboratory environment. The second approach involves research in family-owned animals with cognitive dysfunction syndrome. In this review, we compare the strengths and limitations of housed and family-owned canine models, and appraise their usefulness for deciphering the early mechanisms of AD and developing innovative therapies.

KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer’s disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis.

Putative glucosensing property in rat and human activated microglia.

Microglial cells involved in the pathogenesis of many neurodegenerative diseases acquire the features of cytotoxic and phagocytic cells in response to certain pathogens and inflammatory signals. K(ATP) channels are energy sensors of ATP availability that link the cells metabolic state to its membrane excitability. In pancreatic beta cells, they promote glucose-dependent insulin secretion, and in neurones, hyperpolarization that protects against hypoxic damage. This study analyses activated microglia in an in vivo rat neurodegenerative model based on acute hippocampal glutamate receptor overactivation and in postmortem samples from patients with Alzheimers disease. We demonstrate that in activated microglia the K(ATP) channel components SUR-1 or SUR-2 are present together with glucokinase. Our results indicate that, according to glucose availability, these channels may modify microglia membrane potential. The functional relevance of these channels is seen as a new mechanism modulating the effects of external signals on microglia.