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.

Characterization of a Bioactive Peptide T14 in the Human and Rodent Substantia Nigra: Implications for Neurodegenerative Disease.

The substantia nigra is generally considered to show significant cell loss not only in Parkinson's but also in Alzheimer's disease, conditions that share several neuropathological traits. An interesting feature of this nucleus is that the pars compacta dopaminergic neurons contain acetylcholinesterase (AChE). Independent of its enzymatic role, this protein is released from pars reticulata dendrites, with effects that have been observed in vitro, ex vivo and in vivo. The part of the molecule responsible for these actions has been identified as a 14-mer peptide, T14, cleaved from the AChE C-terminus and acting at an allosteric site on alpha-7 nicotinic receptors, with consequences implicated in neurodegeneration. Here, we show that free T14 is co-localized with tyrosine hydroxylase in rodent pars compacta neurons. In brains with Alzheimer's pathology, the T14 immunoreactivity in these neurons increases in density as their number decreases with the progression of the disease. To explore the functional implications of raised T14 levels in the substantia nigra, the effect of exogenous peptide on electrically evoked neuronal activation was tested in rat brain slices using optical imaging with a voltage-sensitive dye (Di-4-ANEPPS). A significant reduction in the activation response was observed; this was blocked by the cyclized variant of T14, NBP14. In contrast, no such effect of the peptide was seen in the striatum, a region lacking the T14 target, alpha-7 receptors. These findings add to the accumulating evidence that T14 is a key signaling molecule in neurodegenerative disorders and that its antagonist NBP14 has therapeutic potential.

AChE as a spark in the Alzheimer's blaze - Antagonizing effect of a cyclized variant.

The amyloid precursor protein (APP), presenilin 1 (PS1), amyloid beta (Aß), and GSK3 are the effectors, which are significantly associated with progression of Alzheimer's Disease (AD) and its symptoms. A significant protein, acetylcholinesterase (AChE) becomes dysfunctional as a result of cholinergic neuronal loss in AD pathology. However, certain associated peptides potentiate the release of primary neuropathological hallmarks, i.e., senile plaque and neurofibrillary tangles (NFTs), by modulating the alpha 7 acetylcholinesterase receptor (α7nAChR). The AChE variants, T30 and T14 have also been found to be elevated in AD patients and mimic the toxic actions of pathological events in patients. The manuscript discusses the significance of AChE inhibitors in AD therapeutics, by indicating the disastrous role of molecular alterations and elevation of AChE, accompanied with the downstream effects instigated by the peptide, supported by clinical evidence and investigations. The cyclized variant of AChE peptide, NBP14 has been identified as a novel candidate that reverses the harmful effects of T30, T14 and Aß, mainly calcium influx, cell viability and AChE release. The review aims to grab the attention of neuro-researchers towards the significance of triggering effectors in propagating AD and role of AChE in regulating them, which can potentially ace the development of reliable therapeutic candidates, similar to NBP14, to mitigate neurodegeneration.

Antagonism of a key peptide 'T14' driving neurodegeneration: Evaluation of a next generation therapeutic.

T14, a 14mer peptide derived from the C-terminus of acetylcholinesterase (AChE) is a signalling molecule that could drive neurodegeneration via the alpha 7 nicotinic acetylcholine receptor. Its levels increase as Alzheimer's pathology progresses; however, a cyclic variant of the compound, NBP14, can block the effects of the endogenous linear counterpart in-vitro, ex vivo, and in vivo. Here, we explore the antagonistic potential of two 6mer peptides, NBP6A and NBP6B. These are smaller linear versions of NBP14, designed to be more effective by modifying the amino acid residues to enhance receptor blockade alongside other relevant solubility parameters. The peptides were tested in-vitro in PC12 cells on three parameters, calcium influx, cell viability, and AChE release, and ex vivo using voltage sensitive dye imaging (VSDI) in rat brain slices. Neither NBP6A nor NBP6B applied alone had any effect. In PC12 cells, NBP6B was identified as the more potent molecule since it demonstrated more effective blockade of T14 action on calcium influx, cell viability, and AChE release. NBP6B was then further evaluated using VSDI, where it proved twice as potent as NBP14 in blocking the action of T14. The improved effect of NBP6B in blocking the actions of T14, combined with its smaller size suggests that this variant could have even greater therapeutic potential than its original cyclic compound, for treating neurodegenerative disorders.

Antagonising a novel toxin "T14" in Alzheimer's disease: Comparison of receptor blocker versus antibody effects in vitro.

A 14mer peptide, T14, is a possible signaling molecule driving neurodegeneration. Its levels are doubled in the Alzheimer brain, but its effects can be blocked at the target alpha-7 receptor by a cyclised variant, 'NBP14', which has beneficial effects, in a transgenic mouse model, on the behavioral and histochemical profile. Since the antagonism of T14 has evident therapeutic potential, we explore here an alternative method of preventing its action by comparing the efficacy of NBP14 with a proprietorial polyclonal antibody against T14, 'Ab-19', at inhibiting three distinct effects of the peptide in PC12 cells: calcium influx, cell viability and compensatory acetylcholinesterase (AChE) release. None of these three parameters was affected by either blocking agent when applied alone. However, both NBP14 and the Ab-19 exhibited a dose-dependent profile against the actions of T14 in all three scenarios: the least sensitive effect observed was in the lower dose range, for both the antibody and the receptor blocker, in antagonizing T14-triggered release of AChE: this parameter is interpreted as indirect compensation for the T14-induced compromise of cell viability, triggered by the enhanced influx of calcium through the initial binding of the peptide to an allosteric site on the alpha-7 receptor. As such, it is the most delayed and indirect index of T14 action and thus the least relatively impacted by lowest doses of either NBP14 or Ab-19. In all three scenarios however the effects of T14 are successfully offset by either agent and thus offer two potentially very different therapies against Alzheimer's disease.

When a trophic process turns toxic: Alzheimer's disease as an aberrant recapitulation of a developmental mechanism.

Here we review the idea that Alzheimer's disease (AD) results from aberrant activation of a normal developmental mechanism. This process operates in primarily vulnerable, subcortical nuclei with a distinguishing embryological provenance: the basal rather than the alar plate. All cells are dependent for growth on calcium influx yet these neurons retain a sensitivity to trophic factors into maturity. However, as the brain matures this action becomes detrimental such that the trophic process could turn toxic if triggered in adult brain, in retaliation to an initial insult. The signalling molecule driving this trophic-toxic mechanism is a 14mer peptide (T14) that acts on the alpha-7 receptor to enhance calcium entry, inducing excitotoxicity and proliferation of the receptor, perpetuating a feedforward cycle of neurodegeneration including production of beta-amyloid and p-tau. The T14 system has been previously unrecognised as a basic biological process, yet its pharmaceutical manipulation could have valuable clinical applications.

A novel process driving Alzheimer's disease validated in a mouse model: Therapeutic potential.

Introduction: The neuronal mechanism driving Alzheimer's disease (AD) is incompletely understood. Methods: Immunohistochemistry, pharmacology, biochemistry, and behavioral testing are employed in two pathological contexts-AD and a transgenic mouse model-to investigate T14, a 14mer peptide, as a key signaling molecule in the neuropathology. Results: T14 increases in AD brains as the disease progresses and is conspicuous in 5XFAD mice, where its immunoreactivity corresponds to that seen in AD: neurons immunoreactive for T14 in proximity to T14-immunoreactive plaques. NBP14 is a cyclized version of T14, which dose-dependently displaces binding of its linear counterpart to alpha-7 nicotinic receptors in AD brains. In 5XFAD mice, intranasal NBP14 for 14 weeks decreases brain amyloid and restores novel object recognition to that in wild-types. Discussion: These findings indicate that the T14 system, for which the signaling pathway is described here, contributes to the neuropathological process and that NBP14 warrants consideration for its therapeutic potential.