A Novel Peptide Driving Neurodegeneration Appears Exclusively Linked to the α7 Nicotinic Acetylcholine Receptor.

T14, a 14mer peptide, is significantly increased in the pre-symptomatic Alzheimer's disease brain, and growing evidence implies its pivotal role in neurodegeneration. Here, we explore the subsequent intracellular events following binding of T14 to its target α7 nicotinic acetylcholine receptor (nAChR). Specifically, we test how various experimental manipulations of PC12 cells impact T14-induced functional outcomes. Three preparations were compared: (i) undifferentiated vs. NGF-differentiated cells; (ii) cells transfected with an overexpression of the target α7 nAChR vs. wild type cells; (iii) cells transfected with a mutant α7 nAChR containing a mutation in the G protein-binding cluster, vs. cells transfected with an overexpression of the target α7 nAChR, in three functional assays - calcium influx, cell viability, and acetylcholinesterase release. NGF-differentiated PC12 cells were less sensitive than undifferentiated cells to the concentration-dependent T14 treatment, in all the functional assays performed. The overexpression of α7 nAChR in PC12 cells promoted enhanced calcium influx when compared with the wild type PC12 cells. The α7345-348 A mutation effectively abolished the T14-triggered responses across all the readouts observed. The close relationship between T14 and the α7 nAChR was further evidenced in the more physiological preparation of ex vivo rat brain, where T30 increased α7 nAChR mRNA, and finally in human brain post-mortem, where levels of T14 and α7 nAChR exhibited a strong correlation, reflecting the progression of neurodegeneration. Taken together these data would make it hard to account for T14 binding to any other receptor, and thus interception at this binding site would make a very attractive and remarkably specific therapeutic strategy.

A Novel Bioactive Peptide, T14, Selectively Activates mTORC1 Signalling: Therapeutic Implications for Neurodegeneration and Other Rapamycin-Sensitive Applications.

T14 modulates calcium influx via the α-7 nicotinic acetylcholine receptor to regulate cell growth. Inappropriate triggering of this process has been implicated in Alzheimer's disease (AD) and cancer, whereas T14 blockade has proven therapeutic potential in in vitro, ex vivo and in vivo models of these pathologies. Mammalian target of rapamycin complex 1 (mTORC1) is critical for growth, however its hyperactivation is implicated in AD and cancer. T14 is a product of the longer 30mer-T30. Recent work shows that T30 drives neurite growth in the human SH-SY5Y cell line via the mTOR pathway. Here, we demonstrate that T30 induces an increase in mTORC1 in PC12 cells, and ex vivo rat brain slices containing substantia nigra, but not mTORC2. The increase in mTORC1 by T30 in PC12 cells is attenuated by its blocker, NBP14. Moreover, in post-mortem human midbrain, T14 levels correlate significantly with mTORC1. Silencing mTORC1 reverses the effects of T30 on PC12 cells measured via AChE release in undifferentiated PC12 cells, whilst silencing mTORC2 does not. This suggests that T14 acts selectively via mTORC1. T14 blockade offers a preferable alternative to currently available blockers of mTOR as it would enable selective blockade of mTORC1, thereby reducing side effects associated with generalised mTOR blockade.

Cannabinoid 1 Receptor Signaling on Hippocampal GABAergic Neurons Influences Microglial Activity.

Microglia, the resident immune cells of the brain, play important roles in defending the brain against pathogens and supporting neuronal circuit plasticity. Chronic or excessive pro-inflammatory responses of microglia damage neurons, therefore their activity is tightly regulated. Pharmacological and genetic studies revealed that cannabinoid type 1 (CB1) receptor activity influences microglial activity, although microglial CB1 receptor expression is very low and activity-dependent. The CB1 receptor is mainly expressed on neurons in the central nervous system (CNS)-with an especially high level on GABAergic interneurons. Here, we determined whether CB1 signaling on this neuronal cell type plays a role in regulating microglial activity. We compared microglia density, morphology and cytokine expression in wild-type (WT) and GABAergic neuron-specific CB1 knockout mice (GABA/CB1-/-) under control conditions (saline-treatment) and after 3 h, 24 h or repeated lipopolysaccharide (LPS)-treatment. Our results revealed that hippocampal microglia from saline-treated GABA/CB1-/- mice resembled those of LPS-treated WT mice: enhanced density and larger cell bodies, while the size and complexity of their processes was reduced. No further reduction in the size or complexity of microglia branching was detected after LPS-treatment in GABA/CB1-/- mice, suggesting that microglia in naïve GABA/CB1-/- mice were already in an activated state. This result was further supported by correlating the level of microglial tumor necrosis factor α (TNFα) with their size. Acute LPS-treatment elicited in both genotypes similar changes in the expression of pro-inflammatory cytokines (TNFα, interleukin-6 (IL-6) and interleukin 1ß (IL-1ß)). However, TNFα expression was still significantly elevated after repeated LPS-treatment in WT, but not in GABA/CB1-/- mice, indicating a faster development of tolerance to LPS. We also tested the possibility that the altered microglia activity in GABA/CB1-/- mice was due to an altered expression of neuron-glia interaction proteins. Indeed, the level of fractalkine (CX3CL1), a neuronal protein involved in the regulation of microglia, was reduced in hippocampal GABAergic neurons in GABA/CB1-/- mice, suggesting a disturbed neuronal control of microglial activity. Our result suggests that CB1 receptor agonists can modulate microglial activity indirectly, through CB1 receptors on GABAergic neurons. Altogether, we demonstrated that GABAergic neurons, despite their relatively low density in the hippocampus, have a specific role in the regulation of microglial activity and cannabinoid signaling plays an important role in this arrangement.

The effects of orbital frontal cortex damage on the modulation of defensive responses by rats in playful and nonplayful social contexts.

In a series of 3 experiments on rats, 2 hypotheses were tested: (a) that damage to the orbital frontal cortex (OFC) would alter the socially relevant context for executing defensive responses but not their performance and (b) that damage done to the OFC in early infancy would produce more deficits in social behavior than similar damage occurring in adulthood. Bilateral or unilateral OFC damage in adult males did not impair their ability to defend themselves during play fighting and when protecting their food but did impair their ability to modify the pattern of defense in response to different partners. Rats that sustained bilateral damage at 3 days of age not only had deficits in partner-related modulation of defense but also exhibited hyperactivity in their play. The findings thus supported the proposed hypotheses.

Regulatory mechanisms underlying novelty-induced grooming in the laboratory rat.

Bouts of pelage cleaning can be readily evoked in laboratory rodents under conditions of exposure to novelty. Such novelty-induced grooming is described as stereotyped and rostro-caudal in its progression. The patterned structure of novelty-induced grooming makes it particularly attractive for research on the organizational and motivational underpinnings of co-ordinated behaviour. Micro-characteristics of stereotyped novelty-induced grooming bouts were studied in 27 female Wistar rats that were exposed to a novel arena with shelters for a period of 15 min. The order of grooming acts within the initial bouts was rostro-caudal, but subsequent bouts became progressively disorganized in their sequencing. The observed pattern of progressive bout disorganization may be attributed to the gradual dearousal from stress. Differences between consecutive bouts in their micro-characteristics suggest that at least some grooming actions emitted within the context of those bouts operate as relatively independent units of behaviour. Those unitary component actions appear to be integrated into protracted pelage cleaning sequences by a separate mechanism. Similar endogenous mechanisms have been proposed for other co-ordinated motor actions, which suggests that the organizational principles identified in the context of novelty-induced grooming may represent general principles that govern co-ordinated behaviour.