Long-term inhibition of ODC1 in APP/PS1 mice rescues amyloid pathology and switches astrocytes from a reactive to active state

Abstract

Alzheimer's disease (AD) is characterized by the loss of memory due to aggregation of misphosphorylated tau and amyloid beta (Aβ) plaques in the brain, elevated release of inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and reactive oxygen species from astrocytes, and subsequent neurodegeneration. Recently, it was found that enzyme Ornithine Decarboxylase 1 (ODC1) acts as a bridge between the astrocytic urea cycle and the putrescine-to-GABA conversion pathway in the brain of AD mouse models as well as human patients. In this study, we show that the long-term knockdown of astrocytic Odc1 in APP/PS1 animals was sufficient to completely clear Aβ plaques in the hippocampus while simultaneously switching the astrocytes from a detrimental reactive state to a regenerative active state, characterized by proBDNF expression. Our experiments also reveal an effect of astrocytic ODC1 inhibition on the expression of genes involved in synapse pruning and organization, histone modification, apoptotic signaling and protein processing. These genes are previously known to be associated with astrocytic activation and together create a neuroregeneration-supportive environment in the brain. By inhibiting ODC1 for a long period of 3 months in AD mice, we demonstrate that the beneficial amyloid-clearing process of astrocytes can be completely segregated from the systemically harmful astrocytic response to insult. Our study reports an almost complete clearance of Aβ plaques by controlling an endogenous degradation process, which also modifies the astrocytic state to create a regeneration-supportive environment in the brain. These findings present the potential of modulating astrocytic clearance of Aβ as a powerful therapeutic strategy against AD.

Keywords: Alzheimer’s disease; Ornithine decarboxylase 1; Reactive astrocytes.

Fig. 1

Long-term astrocytic KD of ODC1 almost completely clears Aβ plaques in APP/PS1 brains. A Schematic of the astrocytic urea cycle in Alzheimer’s Disease condition (adapted from Ju et al. [4]). B Schematic of the experimental timeline. Red arrows represent time points of virus injection. C Representative images of PyrPeg staining in the hippocampus of virus-injected APP/PS1 animals (Scale bar 200 μm). D Bar graph representing the number of plaques in the hippocampus of virus-injected APP/PS1 animals (each dot represents one brain slice, mouse N = 4–6 in each group). Data represents Mean ± SEM. *p < 0.05, **p < 0.01,***p < 0.001 (Kruskal–Wallis test and Dunn’s multiple comparisons test)

Fig. 2

Long-term ODC1 KD switches astrocytes from reactive to active state in APP/PS1 mice. A Representative images of proBDNF and GFAP-immunoreactive cells in the hippocampus of virus-injected APP/PS1 animals (Scale bar 40 μm, white dashed line represents the interface between CA1 layer lacunosum moleculare and molecular layer of DG). B Bar graph representing the intensity of proBDNF in the GFAP-positive cells from images in panel A (Ordinary one-way ANOVA with Sidak’s multiple comparisons test). C Average number of intersections of the astrocytic branches with concentric circles in Sholl analysis from images in panel A (RM two-way ANOVA with Geisser-Greenhouse correction with Tukey’s multiple comparisons test; *p value for WTScr vs TgScr; #p value for WTScr vs TgODC1; no significant differences between TgScr and TgODC1). D Bar graph representing the ending radius of concentric circles around astrocyte branches in Sholl analysis (Ordinary one-way ANOVA with Tukey’s multiple comparisons test). E Bar graph representing the sum of intersections made by the astrocyte branches and concentric circles in Sholl analysis (Ordinary one-way ANOVA with Tukey’s multiple comparisons test). Data represents Mean ± SEM. # or*p < 0.05, ## or **p < 0.01,### or ***p < 0.001, #### or ****p < 0.0001

Fig. 3

Transcriptome of long-term ODC1-inhibited astrocytes shows dramatic changes, resembling enriched environment-house animal brains. A Experimental timeline schematic for bulk RNA sequencing of primary astrocyte culture. B Volcano plot representing the differential expression of specific genes associated with astrocyte reactivity, learning and memory, apoptosis, transcriptional regulation, ubiquitination and autophagy from bulk RNA-Seq. Horizontal dotted line refers to p < 0.05, vertical dotted line indicates no change in expression level). C Volcano plots representing the differential expression of genes involved in different GO terms from the bulk-RNASeq dataset. Horizontal dotted line refers to p < 0.05, vertical dotted line indicates no change in expression level. D Gene ontology analysis for synapse-associated genes from bulk RNA-Seq of Aβ and Aβ + DFMO-treated primary cultured astrocytes. E Gene ontology analysis for histone modification-associated genes from bulk RNA-Seq of Aβ and Aβ + DFMO-treated primary cultured astrocytes. F Gene ontology analysis for protein processing pathways and related genes from bulk RNA-Seq of Aβ and Aβ + DFMO-treated primary cultured astrocytes. G Gene ontology analysis for genes associated with apoptosis from bulk RNA-Seq of Aβ and Aβ + DFMO-treated primary cultured astrocytes

Fig. 4

Long-term inhibition of ODC1 in AD creates an Aβ-clearing and neuro-supportive active state. Top-Left: In AD, astrocytes show upregulated autophagy to uptake accumulated amyloid deposits and switch-on the urea cycle along with upregulation of enzyme ODC1, which increases putrescine, GABA, H₂O₂ and ammonia production, causing neurodegeneration and memory impairment. Top-Right: Short-term ODC1 inhibition in AD mouse models blocks the conversion of ornithine to putrescine, increases the flux of the urea cycle, reduces production of putrescine, GABA, H₂O₂ and thereby preventing oxidative stress, neurodegeneration, and memory impairment. These astrocytes can be called Aβ-detoxifying astrocytes (schematics adapted from Ju et al. [4]) Bottom: Long-term inhibition of ODC1 in AD-like astrocytes can improve Aβ plaque clearance and drastically alter the transcriptome of astrocytes, by increasing histone modifications, Nrf2 expression, proBDNF production, protein-associated processes and cellular metabolism. These changes, along with reduced apoptotic signaling, ubiquitination and synaptic pruning-associated genes, resemble the transcriptome of EE active astrocytes and can create a neuro-supportive environment for regeneration (Created using biorender.com)