Methylene blue inhibits Caspase-6 activity, and reverses Caspase-6-induced cognitive impairment and neuroinflammation in aged mice

Libin Zhou,Libin Zhou,P Jesper Sjöström,P Jesper Sjöström,Olivier Beauchet,Olivier Beauchet,Anastasia Noël,Joseph Flores,Andrea C Leblanc

doi:10.1186/s40478-019-0856-6

Abstract

Activated Caspase-6 (Casp6) is associated with age-dependent cognitive impairment and Alzheimer disease (AD). Mice expressing human Caspase-6 in hippocampal CA1 neurons develop age-dependent cognitive deficits, neurodegeneration and neuroinflammation. This study assessed if methylene blue (MB), a phenothiazine that inhibits caspases, alters Caspase-6-induced neurodegeneration and cognitive impairment in mice. Aged cognitively impaired Casp6-overexpressing mice were treated with methylene blue in drinking water for 1 month. Methylene blue treatment did not alter Caspase-6 levels, assessed by RT-PCR, western blot and immunohistochemistry, but inhibited fluorescently-labelled Caspase-6 activity in acute brain slice intact neurons. Methylene blue treatment rescued Caspase-6-induced episodic and spatial memory deficits measured by novel object recognition and Barnes maze, respectively. Methylene blue improved synaptic function of hippocampal CA1 neurons since theta-burst long-term potentiation (LTP), measured by field excitatory postsynaptic potentials (fEPSPs) in acute brain slices, was successfully induced in the Schaffer collateral-CA1 pathway in methylene blue-treated, but not in vehicle-treated, Caspase-6 mice. Increased neuroinflammation, measured by ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia numbers and subtypes, and glial fibrillary acidic protein (GFAP)-positive astrocytes, were decreased by methylene blue treatment. Therefore, methylene blue reverses Caspase-6-induced cognitive deficits by inhibiting Caspase-6, and Caspase-6-mediated neurodegeneration and neuroinflammation. Our results indicate that Caspase-6-mediated damage is reversible months after the onset of cognitive deficits and suggest that methylene blue could benefit Alzheimer disease patients by reversing Caspase-6-mediated cognitive decline.

Highlights

Alzheimer disease (AD) is defined by the appearance of age-dependent progressive cognitive impairment, brain amyloid beta peptide (Aβ)laden extracellular plaques, intraneuronal aggregated Tau protein-containing neurofibrillary tangles (NFT), synaptic loss, and neuroinflammation
This study demonstrates that Casp6-induced cognitive deficits, neuritic degeneration, and neuroinflammation are reversible and implies that human Casp6-dependent cognitive decline in AD may be prevented with a Casp6 inhibitor
Validation of human Caspase-6 gene expression in the Knock in (KI)/Cre mice Validation of the expression of CASP6 was done after behavioral assessments upon sacrifice of the mice but we present it first because of unexpected findings as described below

Summary

Introduction

AD is defined by the appearance of age-dependent progressive cognitive impairment, brain amyloid beta peptide (Aβ)laden extracellular plaques, intraneuronal aggregated Tau protein-containing neurofibrillary tangles (NFT), synaptic loss, and neuroinflammation. In aged non-cognitively impaired (NCI) individuals, active Casp is observed in entorhinal cortex (ERC) and hippocampal CA1 neurons [6, 20, 39], the first areas presenting NFT in AD [8]. In these individuals, higher levels of Casp activity correlate with lower performance in episodic and semantic memory, the two types of memory first affected in AD [39]

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