Astrocytic K⁺ clearance during disease progression in amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which patients lose motor functions due to progressive loss of motor neurons in the cortex, brainstem, and spinal cord. Whilst the loss of neurons is central to the disease, it is becoming clear that glia, specifically astrocytes, contribute to the onset and progression of neurodegeneration. Astrocytes play an important role in maintaining ion homeostasis in the extracellular milieu and regulate multiple brain functions by altering their extracellular concentrations. In this study, we have investigated the ability of astrocytes to maintain K + homeostasis in the brain via direct measurement of the astrocytic K + clearance rate in the motor and somatosensory cortices of an ALS mouse model (SOD1 G93A ). Using electrophysiological recordings from acute brain slices, we show region-specific alterations in the K + clearance rate, which was significantly reduced in the primary motor cortex but not the somatosensory cortex. This decrease was accompanied by significant changes in astrocytic morphology, impaired conductivity via Kir4.1 channels and low coupling ratio in astrocytic networks in the motor cortex, which affected their ability to form the K + gradient needed to disperse K + through the astrocytic syncytium.These findings indicate that the supportive function astrocytes typically provide to motoneurons is diminished during disease progression and provides a potential explanation for the increased vulnerability of motoneurons in ALS.

Potassium homeostasis during disease progression of Alzheimer’s Disease

“…Various KCl concentrations were applied to brain slices at a constant distance of ~10 μm from the K + -selective microelectrode through a puffing pipette (tip diameter of 1μm) for 0.1 seconds at 10 psi, as previously described (34,35). The rate constant ( = 1/) and decay time (defined as the time it took the amplitude to decay from 90-10%) were used for statistical analysis.…”

“…To assess astrocytic coupling, individual astrocytes were loaded with an intracellular solution containing biocytin (0.3%; Sigma) for 12 min in a whole-cell mode, as previously described (7,34,35). Hyperpolarizing step currents (50 pA for 500 ms at 0.5 Hz) were applied via the recording electrode to facilitate the spreading of the biocytin to neighbouring astrocytes, as previously described.…”

“…2E-F), indicating that astrocytic reactivity was region-specific and not uniform across all brain regions. We next assessed astrocytic connectivity via intracellular injections of 0.3% biocytin and evaluation of the number of primary coupled astrocytes to the patched astrocytes as previously described (7,34,35). Our data indicate a significant decrease in the number of primary coupled astrocytes in the hippocampus of 5xFAD mice, which was due to the 5xFAD mutations (F(1, 15)=7.33, p<0.01 for the factor '5xFAD mutations'; F(1, 15) = 0.14, p=0.7 for the factor 'aging' and F(1, 15) = 1.57, p = 0.22 for the factor 'Interaction', two-way ANOVA).…”

“…To assess the mechanisms underlying the altered clearance rate, we next investigated specific cellular and intracellular processes that impact the clearance rate, including K + uptake mediated via inward rectifying K + (Kir) channels and K + spatial buffering via the astrocytic syncytium (8,41,42). Within the K + inward rectifying channel family, Kir4.1 channels are specifically expressed in astrocytes and oligodendrocytes and are responsible for the majority of K + uptake following neuronal activity, reviewed by (35,43,44). Currently, there is no specific blocker for Kir4.1 channels, however previous studies have shown that a low concentration of BaCl2 (100 μM) can selectively block Kir4.1 channels and thus impair the K + uptake mechanism in astrocytes (7,34,37,(45)(46)(47).…”

“…These concentrations were elected as they are correlated to different levels of network activity and involves different phases within the astrocytic clearance mechanisms(7,8). The [K + ]o clearance rate constant (λ = 1/τ) was calculated from the fitted exponential decline of the voltage signal (90%-10%) using a leastsquares fitting routine (Chebyshev algorithm Clampfit 10, Molecular devices), as previously described(35).To assess the impact of 'ageing', '5xFAD mutations' and 'K + concentration' on the clearance rate, we performed a three-way ANOVA analysis on these variables. Our results indicate that the clearance rate recorded in the hippocampus was significantly affected by all three factors (F(1, 149) = 70.89, p < 0.0001 for the factor 'aging'; F(1, 149) = 34.17, p < 0.0001 for the factor '5xFAD mutations' and F(2, 149) = 39.25, p < 0.001 for the factor 'K + concentration'), with significant two-way interaction between 'ageing x 5xFAD mutations' (F(1, 149) = 20.62, p < 0.001), and three-way interaction between all factors (F(2, 149) = 6.266, p < 0.005, three-way ANOVA, Figure3C).…”