Introduction
Astrocytic calcium signaling is disrupted at early stages of Alzheimer’s disease (AD), before amyloid plaque deposition. This early dysfunction mediates neuronal and network disturbances, positioning astrocytes in a central role in disease progression¹. Because astrocytic calcium dynamics support key regulatory functions in the brain, identifying the mechanisms that initiate this disruption is critical. Lysosomes, in addition to their degradative role, serve as intracellular calcium stores that regulate organelle crosstalk, positioning them to shape astrocytic signaling and rendering them vulnerable to AD-related stress. Here, we investigate whether lysosomal dysfunction emerges before amyloid deposition and whether it drives early astrocytic calcium signaling deficits in vivo.

Methods

In-vivo two-photon calcium imaging was performed in heterozygous App^NL-G-F knock-in mice² and wild-type littermates at pre- and post-plaque stages following astrocyte-specific AAV expression of cytosolic or lysosome-targeted calcium indicators. Imaging experiments typically included N=4 mice per group, with 25-48 astrocytes analyzed per mouse. Lysosomal acidity and protein expression were assessed using the ratiometric pH-sensitive FIREpHLy probe and immunohistochemistry, respectively, while amyloid pathology was quantified using 82E1 staining. Statistical analyses employed ART ANOVA or nested t-tests with multiple-comparison correction as appropriate. All experimental procedures complied with the UK Animals (Scientific Procedures) Act and were approved by the relevant local ethical review board.

Results

In-vivo two-photon imaging revealed a pronounced suppression of astrocytic cytosolic calcium signaling at pre-plaque stages of AD, coinciding with early abnormalities in lysosomal function. Astrocytic lysosomes exhibited reduced luminal acidity, altered expression of lysosomal markers, and a marked loss of spontaneous lysosomal calcium release. This deficit was associated with reduced expression of the lysosomal calcium channel TRPML1. Astrocyte-specific restoration of TRPML1 rescued lysosomal calcium release and normalized cytosolic calcium signaling at early stages. Importantly, early TRPML1 expression prevented the later emergence of astrocytic calcium hyperactivity, reduced astrocyte reactivity, and markedly attenuated amyloid plaque accumulation in vivo.

Discussion and conclusion

These findings identify impaired lysosomal calcium release as an early upstream driver of astrocytic calcium signaling deficits in Alzheimer’s disease. Our data link early lysosomal dysfunction to a broader collapse of astrocytic calcium signaling and subsequent disease progression. Restoring lysosomal calcium release stabilized astrocytic calcium dynamics, prevented later astrocytic hyperactivity, and reduced amyloid pathology. Together, this work positions astrocytic lysosomal calcium signaling as a central and tractable mechanism in early AD.