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Photoactivated adenylyl cyclase in cortical astrocytes promotes synaptic potentiation and reveals alterations in Huntington’s Disease

ABSTRACT: Astrocytes are glial cells essential for neuronal plasticity. However, the role of astrocytic cAMP signalling in brain activity and function is still missing. We expressed DdPAC, a photoactivated adenylyl cyclase that increases cAMP upon red light, in cortical astrocytes to investigate its effects. We assessed synaptic transmission using multi-electrode arrays, and molecular changes through proteomic, phosphoproteomic, and metabolic analyses. We also measured brain hemodynamics and analysed motor behaviour in wild-type (WT) and R6/1 Huntington's disease (HD) mice. DdPAC stimulation in cortical astrocytes induced long-term synaptic potentiation in WT mice, which was PKA and NMDAR-dependent but Ca2+-independent. It also triggered PKA-signalling pathways and modulated synaptic and morphology-associated proteins. Further, cAMP signalling increased cortical blood flow and improved motor learning in WT mice but show differential responses in HD mice. Our results unravel the impact of cAMP signalling in astrocytes to synaptic to brain function, and highlight its alterations in HD.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Astrocyte Of The Cerebral Cortex, Astroglial Cell

DISEASE(S): Huntington Disease

SUBMITTER: Enrique Santamaría

LAB HEAD: Mercè Masana

PROVIDER: PXD054635 | Pride | 2025-09-18

REPOSITORIES: Pride

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Project description:Huntingtons disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat mutation in the Huntingtin (HTT) gene. The mutation impacts neuronal protein homeostasis and cortical/striatal circuitry. SUMOylation, a post translational modification with broad cellular effects, directly modifies the Huntingtin protein (HTT), along with other key neuronal and synaptic proteins. Here we investigated proteome wide changes in striatal protein SUMOylation/protein SUMO interactions in the context of HD using R6/2 transgenic and non-transgenic (NT) control mice by performing SUMO protein enrichment from tissue followed by mass spectrometry. Significant changes in enrichment of known and previously unknown SUMOylated or SUMO interacting proteins were observed including those involved in presynaptic function and cytomatrix at the active zone scaffolding, cytoskeleton organization, and glutamatergic signaling. A network based approach identified altered pathways in HD tissue to include clathrin mediated endocytosis signaling, synaptogenesis signaling, synaptic long term potentiation, and SNARE signaling. Furthermore, the metabotropic glutamate receptor 7 (mGluR7), a key player in glutamatergic signaling, a core signaling pathway disrupted in HD was SUMO enriched and we show SUMO modification is enhanced by the E3 SUMO ligase Protein Inhibitor of Activated STAT1 (PIAS1). To evaluate functional measures of neuronal activity in HD cells in vitro we utilized primary neuronal cultures from R6/2 and NT mice and evaluated how modulation of SUMOylation via reduction of PIAS1 may impact specific readouts. A receptor internalization assay showed decreased mGluR7 internalization in R6/2 neurons compared to NT, and siRNA mediated knockdown of PIAS1 prevented this HD phenotype. In addition, microelectrode array analysis on primary neuron cultures indicated early timepoint hyperactivity in HD cells, while later timepoints demonstrated deficits in several measurements of neuronal activity within cortical neurons. HD phenotypes were rescued at select timepoints following knockdown of PIAS1. Taken together our results provide a mouse brain SUMO ome resource and show that significant alterations occur within the post-translational landscape and SUMO protein interactions for synaptic proteins in HD.

Project description:Alzheimer’s disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, how this metabolic interplay may be affected during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aβ accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in hippocampal brain slices of 5xFAD mice. This hyperactive neuronal phenotype coincided with decreased hippocampal TCA cycle metabolism mapped by stable 13C isotope tracing. Particularly, reduced astrocyte TCA cycle activity led to decreased glutamine synthesis, in turn hampering neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, cerebral cortical slices of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism suggesting a metabolic compensation. When we explored the brain proteome and metabolome of the 5xFAD mice, we found limited changes, supporting that the functional metabolic disturbances between neurons and astrocytes are early events in AD pathology. In addition, we show that synaptic mitochondrial and glycolytic function was impaired selectively in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex region and cell specific metabolic adaptations, in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunction in AD.

			Action	DRS
	20240422_175234_311-MerceMassana_phospho.sne	Other
	Phospho_LSR01.raw	Raw
	Phospho_LSR02.raw	Raw
	Phospho_LSR03.raw	Raw
	Phospho_LSR04.raw	Raw

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Photoactivated adenylyl cyclase in cortical astrocytes promotes synaptic potentiation and reveals alterations in Huntington’s Disease

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