Authors :
Presenting Author: Kieran Normoyle, MD PhD – Massachusetts General Hospital / Harvard Medical School
Volodymyr Dzhala, PhD – Massachusetts General Hospital / Harvard Medical School
Kyle Lillis, PhD – Massachusetts General Hospital
Kiyoshi Egawa, MD PhD – Hokkaido University
Kevin Staley, MD – Massachusetts General Hospital
Rationale:
The reversal potential of GABAA receptors (EGABA) is dependent upon the chloride concentrations on both sides of the neuronal membrane. We recently discovered that the extracellular chloride concentration [Cl-]o is non-uniform and roughly only half the chloride in bulk cerebrospinal fluid. We also observed that removal of a portion of the immobile polyanions from the extracellular space (ECS), mimicking injury-induced metalloprotease (MMP) activation, results in a shift toward higher local [Cl-]o. Methods:
We used 2-photon Fluorescence Lifetime Imaging (FLIM) of a custom chloride-sensitive fluorophore constrained to the ECS by conjugation with 10 kilodalton dextran and 2-photon photolysis of single neurons to model acute brain injuries, first in organotypic cultures of hippocampal slices and then in vivo via mouse cortical window implantation in wildtype and APP/PS1 Alzheimer’s disease mouse models. We used slice injury as well as 2-photon photolysis of single neurons to model acute brain injuries. Intraneuronal chloride was measured with the ratiometric reporter Super Chlomeleon. Amyloid beta (Ab) aggregate accumulations were identified with near-infrared fluorescent stain CRANAD-3. Results:
Having discovered that the [Cl-]o between neurons both in vitro and in vivo is only about half that of bulk CSF chloride, and that digestion of a prominent immobile extracellular anion in the brain (chondroitin sulfate) leads to release of these sulfated moieties and a shift to higher chloride concentrations, we next asked what would happen to [Cl-]o when the sulfate moieties of the matrix are freed by endogenous MMPs after brain injury.
We found a strong dependence of [Cl-]o vs distance from photolytic injury, with [Cl-]o increasing to ACSF levels in proximity to photolysed neurons, which we hypothesize is due to mobilization of immobile extracellular anions following MMP release and activation. We then tested whether excess immobile extracellular anions lead to low [Cl-]o. Using 19mo APP/PS1 mice we measured [Cl-]o at sites of polyanionic Ab aggregates that accumulate in ECS in Alzheimer’s disease. We observed lower [Cl-]o in proximity to Ab aggregate accumulations. These changes in [Cl-]o should also alter the neuronal intracellular chloride ([Cl-]i) via the activity of the high-velocity equilibrative membrane chloride transporters. In adolescent wildtype mice we have observed increased [Cl-]i upon acute injury and subsequent [Cl-]o increase.
Conclusions:
Taken together these data confirm that displacement of [Cl-]o by Donnan forces is an underappreciated mechanism by which [Cl-]o may become pathologically high by degradation of extracellular polyanions or pathologically low due to accumulations of polyanions, such as Ab aggregates. [Cl-]o is partially displaced by carboxylates and sulfates in the extracellular matrix. Pathophysiological removal or deposition of extracellular immobile anions alters the distribution of chloride in both the extra- and intracellular spaces. These findings have immediate implications for the treatment of cytotoxic edema, seizures, and neurodegenerative disorders including Alzheimer’s disease.
Funding:
R35NS116852