Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging (2022)
Figure 6

Figure 6. XBP1s gene delivery to aged hippocampus reverses age-associated phenotypes at the morphological and electrophysiological levels.

(A) Basal (-) and picrotoxin-induced (+) of firing rates (Mean and s.e.m.) were measured in hippocampal brain slices from aged mice (24 months) injected with AAV2-Mock or AAV2-XBP1s (n = 633-832 neurons; n = 6, 9 animals, respectively; Kolmogorov-Smirnov test).

(B) Basal (-) and picrotoxin-induced (+) burst activity (Mean and s.e.m.) were measured in Hippocampal brain slices from aged mice injected with AAV2-Mock or AAV2-XBP1s (n = 832, 644 neurons from 3-4 animals, Kolmogorov-Smirnov test).

(C) Left panel: Mean and s.e.m. of field excitatory postsynaptic potentials amplitudes in brain slices derived from aged animals injected with AAV2-Mock or AAV2-XBP1s (24 months). Recordings of PEPs following 1 h after theta burst stimulus (TBS) to induce LTP. Right panel: Mean and s.e.m. of PEPs in the last ten minutes following TBS. n = 6, 9 animals, respectively; n = 17-28 slices/animal, unpaired Student's t test).

(D) Left panel: Representative images showing dendritic spines of aged (22-24 months) animals injected with AAV2-eGFP (mock) or with AAV2-XBP1s (arrows indicate dendritic spines). Young animal dendrite is shown for reference. 60x magnification. Scale bar: 5μm. Right panel: Mean and s.e.m. of spine density per μm for indicated experimental groups (n = 24, 53 and 35 dendrites from 3, 5 and 6 animals, respectively; unpaired Student's t test.

(E) Left panel: Representative images of β-galactosidase staining of hippocampal slices derived from aged mice injected with AAV2-Mock or AAV2-XBP1s (arrow indicate β-galactosidase staining). Magnification 20x; scale bar, 200 μm. Right panel: Histograms show mean and s.e.m. of percentage of β-galactosidase-stained area. n = 6, 6; unpaired Student's t test.

(F) Left panel: Immunofluorescence of γH2aX (red) counterstained with nuclei (blue) comparing aged mice injected with AAV-Mock or AAV-XBP1s. Magnification: 40x; Scale bar: 50μm. Right panel: Histograms show mean and s.e.m. of percentage of γH2A- positive cells; unpaired Student's t test comparing the aged groups ( n = 6, 6 animals/group; unpaired Student's t test).

(G) Following LC-MS/MS analysis of the hippocampal proteome, gene set enrichment analysis was performed using EnrichR with Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Graphs depict most enriched terms based on combined scored computed by EnrichR platform comparing aged mice injected with AAV-Mock or AAV-XBP1s. Terms were divided in two graphs related to synaptic function or neurodegeneration.

(H) Volcano plot for proteomic comparison of hippocampal tissue derived from aged animals injected with AAV-Mock or AAV-XBP1s. FDR (y-axis) and fold-change (log2) (x-axis) are indicated. Dotted lines delineate cut-off used to filter genes for functional enrichment analysis. Indicated genes associated with each enriched terms connected to synaptic function, neurodegeneration, metabolism and ECM-receptor interaction are colored.

Data information: Graphs indicate P values with the following legend: * = P < 0.05 ; ** = P <0.01; ***: P <0.005; **** = P < 0.001.

Table 1: Significantly altered genes in RNA-seq comparing the hippocampus of demented patients with controls (fold change > 1.2) in the Aging, Dfementia and Traumatic injury Study database. Most altered genes compose a functional network of interactions related to unfolded protein binding and processing. Table indicates gene symbols, fold changes, p Values and enriched terms in gene ontology and Kyoto Encyclopedia of Genes and Genomes databases:

Table 2: Most enriched terms in KEGG database comparing mice in different ages. Table indicates terms, associated genes and combined score computed in EnrichR platform.

Table 3: Most enriched terms in KEGG database comparing TgXBP1s with age-matched WT mice. Table indicates enriched terms and their associated genes. Symbols highlight when those terms are enriched in TgXBP1s animals within each age-comparison.

Enriched Terms

Synaptic function

Synaptic vesicle cycle *

CLTB;CLTA;SLC6A11;SLC17A7; SLC1A3

Long-term potentiation*

GRIA1;PPP3CA;GRIN2A;RAP1B;CAMK2D;PPP3R1;ADCY1

Glutamatergic synapse *

GRIA1;PPP3CA;GRIN2A;SLC17A7;PPP3R1;SLC1A3;ADCY1

Calcium signaling pathway †

Dopaminergic synapse *

GRIA1;PPP3CA;GRIN2A; CAMK2D

Endocytosis †

ARPC1A;CLTB;CLTA;ARPC4;WIPF3;HSPA1A; WIPF3;HSPA1A

Long-term depression ‡

cAMP signaling pathway †

GRIA1;GRIN2A;RAP1B;CAMK2D;ADCY1; ATP2B4

GABAergic synapse *

SLC6A11;ADCY1

Neuroactive ligand-receptor †

MAPK pathway †

PPP3CA;CACNG8;TAOK3;STMN1;RAP1B;PPP3R1; TAOK3; HSPA1A

Axon guidance †

PPP3CA;CAMK2D;PPP3R1;PTPN11

Neurodegeneration

ALS *

GRIA1;PPP3CA;GRIN2A;PPP3R1;NEFL;NEFM;NEFH

Prion diseases †

Alzheimer disease *

PPP3CA;GRIN2A;PPP3R1

Apoptosis **

LMNA;LMNB2;LMNA;LMNB2

Necroptosis †

HIST3H2A;H2AFY;CAMK2D;HIST3H2A;H2AFX

Cellular senescence *

PPP3CA;PPP3R1

Metabolism/Bioenergetics

Glucagon signaling pathway *

Glycolysis / Gluconeogenesis ‡

LDHB

GnRH signaling pathway *

PTK2B;CAMK2D;ADCY1

Other Terms

Spliceosome **

HNRNPM;DDX5;HNRNPA3;HNRNPK;SRSF1;SRSF2;HNRNPU;SRSF4;HNRNPC;PRPF19; HSPA1A

Ribosome *

RPLP2;RPS13;RPL18

* Term enriched in Young and Middle Aged TgXBP1s

‡ Term enriched only Young TgXBP1s

† Term enriched in Young, Middle Aged and Aged TgXBP1s

** Term enriched in Young and Aged TgXBP1s

Table 4: Table depicts most enriched terms in KEGG database comparing aged mice injected with AAV-Mock or AAV-XBP1s. Table indicates enriched terms and their associated genes. Symbols highlight whether enriched terms correlate with terms found during the natural course of aging in control animals.

APP;PPP3R1;NDUFV2;

CALM2

* Term associated to Young WT x Aged WT

‡ Term associated to Young WT x Middle Aged WT

† Term associated to Middle Aged WT x Aged WT