Combination of Endothelin-1 (ET-1) and L-NAME to Induce Murine Focal Cortical Stroke with Persistent Sensorimotor Deficits.

The endothelin-1 (ET-1) model of stroke involves the stereotactic injection of the vasoconstrictor ET-1 to produce a focal ischemic injury. In rats, this model produces consistent deficits, in contrast to more variable results in mice. In this chapter, we describe a new method to induce a murine focal ischemic cortical stroke by injecting L-NAME, another potent vasoconstrictor , in combination with ET-1 into the sensorimotor cortex. This ET-1 /L-NAME stroke induction protocol produces consistent focal cortical infarcts and sensorimotor functional impairments in C57BL/6 mice.

AgRP neuronal activity across feeding-related behaviours.

AgRP neurons trigger one of the most potent orexigenic responses and are both necessary and sufficient for feeding. Recent technical advances for monitoring in vivo neuronal activity have revisited a previously well-established model of AgRP neurons' feeding regulatory effects. Our current understanding of AgRP neurons has increased in complexity and revealed a fine-tuned regulation of their activity dynamics across the whole sequence of feeding-related behaviours. This review focuses on recent studies that refined and re-evaluated our understanding of the regulatory principles and behavioural effects of AgRP circuits. We aim to cover major discoveries on the dynamic regulation of AgRP neuronal activity by exteroceptive and interoceptive food-related cues, their pleiotropic effects in feeding and whole-body homeostasis, and the associated AgRP circuits. The function and regulation of AgRP neuron will be sequentially discussed across the temporal series of behavioural and physiological changes occurring during the appetitive (food craving and foraging), the anticipatory (discovery of food-predicting cues), and the consummatory/post-ingestive phase of feeding (calorie ingestion).

Metformin promotes CNS remyelination and improves social interaction following focal demyelination through CBP Ser436 phosphorylation.

Individuals with demyelinating diseases often experience difficulties during social interactions that are not well studied in preclinical models. Here, we describe a novel juvenile focal corpus callosum demyelination murine model exhibiting a social interaction deficit. Using this preclinical murine demyelination model, we discover that application of metformin, an FDA-approved drug, in this model promotes oligodendrocyte regeneration and remyelination and improves the social interaction. This beneficial effect of metformin acts through stimulating Ser436 phosphorylation in CBP, a histone acetyltransferase. In addition, we found that metformin acts through two distinct molecular pathways to enhance oligodendrocyte precursor (OPC) proliferation and differentiation, respectively. Metformin enhances OPC proliferation through early-stage autophagy inhibition, while metformin promotes OPC differentiation into mature oligodendrocytes through activating CBP Ser436 phosphorylation. In summary, we identify that metformin is a promising remyelinating agent to improve juvenile demyelination-associated social interaction deficits by promoting oligodendrocyte regeneration and remyelination.

Ectopic expression of aPKC-mediated phosphorylation in p300 modulates hippocampal neurogenesis, CREB binding and fear memory differently with age.

Epigenetic modifications have become an emerging interface that links extrinsic signals to alterations of gene expression that determine cell identity and function. However, direct signaling that regulates epigenetic modifications is unknown. Our previous work demonstrated that phosphorylation of CBP at Ser 436 by atypical protein kinase C (aPKC) regulates age-dependent hippocampal neurogenesis and memory. p300, a close family member of CBP, lacks the aPKC-mediated phosphorylation found in CBP. Here, we use a phosphorylation-competent p300 (G442S) knock-in (KI) mouse model that ectopically expresses p300 phosphorylation in a homologous site to CBP Ser436, and assess its roles in modulating hippocampal neurogenesis, CREB binding ability, and fear memory. Young adult (3 months) p300G422S-KI mice exhibit enhanced hippocampal neurogenesis due to increased cell survival of newly-generated neurons, without alterations in CREB binding and contextual fear memory. On the other hand, mature adult (6 months) p300G422S-KI mice display reduced CREB binding, associated with impaired contextual fear memory without alterations in hippocampal neurogenesis. Additionally, we show that repulsive interaction between pS133-CREB and pS422-p300G422S may contribute to the reduced CREB binding to p300G422S. Together, these data suggest that a single phosphorylation change in p300 has the capability to modulate hippocampal neurogenesis, CREB binding, and associative fear memory.

The aPKC-CBP Pathway Regulates Post-stroke Neurovascular Remodeling and Functional Recovery.

Epigenetic modifications have emerged as attractive molecular substrates that integrate extrinsic changes into the determination of cell identity. Since stroke-related brain damage releases micro-environmental cues, we examined the role of a signaling-induced epigenetic pathway, an atypical protein kinase C (aPKC)-mediated phosphorylation of CREB-binding protein (CBP), in post-stroke neurovascular remodeling. Using a knockin mouse strain (CbpS436A) where the aPKC-CBP pathway was defective, we show that disruption of the aPKC-CBP pathway in a murine focal ischemic stroke model increases the reprogramming efficiency of ischemia-activated pericytes (i-pericytes) to neural precursors. As a consequence of enhanced cellular reprogramming, CbpS436A mice show an increased transient population of locally derived neural precursors after stroke, while displaying a reduced number of i-pericytes, impaired vascular remodeling, and perturbed motor recovery during the chronic phase of stroke. Together, this study elucidates the role of the aPKC-CBP pathway in modulating neurovascular remodeling and functional recovery following focal ischemic stroke.

Extracellular cytochrome c as an intercellular signaling molecule regulating microglial functions.

BACKGROUND: Cytochrome c is well known to be released from mitochondria into the cytosol where it can initiate apoptosis. Recent studies indicate that cytochrome c is also released into the extracellular space by both healthy and damaged cells, where its function is not well understood. We hypothesized that extracellular cytochrome c could function as an intercellular signaling molecule of the brain, which is recognized by brain microglia. These cells belong to the mononuclear phagocyte system and can be activated by endogenous substances associated with diverse pathologies including trauma, ischemic damage and neurodegenerative diseases. METHODS: Three different cell types were used to model microglia. Respiratory burst activity, nitric oxide production and cytotoxic secretions were measured following exposure of microglial cells to cytochrome c. RESULTS: We showed that extracellular cytochrome c primed the respiratory burst response of differentiated HL-60 cells, enhanced nitric oxide secretion by BV-2 cells, and augmented cytotoxicity of differentiated THP-1 cells. We demonstrated that the effects of cytochrome c on microglia-like cells were at least partially mediated by the toll-like receptor 4 (TLR4) and c-Jun N-terminal kinases (JNK) signaling pathway. CONCLUSIONS: Extracellular cytochrome c can interact with microglia TLR4 and modulate select functions of these brain immune cells. GENERAL SIGNIFICANCE: Our data identifies extracellular cytochrome c as a potential intercellular signaling molecule, which may be recognized by microglia causing or enhancing their immune activation. The data obtained support targeting TLR4 and JNK signaling as potential treatment strategies for brain diseases characterized by excessive cellular death and activation of microglia.

The aPKC-CBP Pathway Regulates Adult Hippocampal Neurogenesis in an Age-Dependent Manner.

While epigenetic modifications have emerged as attractive substrates to integrate environmental changes into the determination of cell identity and function, specific signals that directly activate these epigenetic modifications remain unknown. Here, we examine the role of atypical protein kinase C (aPKC)-mediated Ser436 phosphorylation of CBP, a histone acetyltransferase, in adult hippocampal neurogenesis and memory. Using a knockin mouse strain (CbpS436A) in which the aPKC-CBP pathway is deficient, we observe impaired hippocampal neuronal differentiation, maturation, and memory and diminished binding of CBP to CREB in 6-month-old CbpS436A mice, but not at 3 months of age. Importantly, elevation of CREB activity rescues these deficits, and CREB activity is reduced whereas aPKC activity is increased in the murine hippocampus as they age from 3 to 6 months regardless of genotype. Thus, the aPKC-CBP pathway is a homeostatic compensatory mechanism that modulates hippocampal neurogenesis and memory in an age-dependent manner in response to reduced CREB activity.