Fig 2: Neuropathogenesis in ADEV injected mice: Representative WB images of different brain regions demonstrating the expression of (a) AβmOC64, (b) APP, (c) pTau. Data are presented as mean ± SEM. One‐way ANOVA followed by Bonferroni post hoc test was performed, *p < 0.05 versus CEV, #p < 0.05 versus TEV. n = 3 (CEV, HCEV), 4 (TEV, HTEV) mice/group. APP, amyloid precursor protein; CER, cerebellum; CEV, ADEVs from scrambled siRNA transfected HPAs; FC, Frontal cortex; GAPDH, Glyceraldehyde‐3 phosphate dehydrogenase; HC, Hippocampus; HCEV, ADEVs from HIF‐1α siRNA transfected HPAs, HTEV, ADEVs from HIF‐1α siRNA transfected Tat exposed HPAs; OC, Occipital cortex; PC: Parietal cortex; STR, Striatum; TEV, ADEVs from scrambled siRNA transfected Tat exposed HPAs.

Fig 3: Characterization of astrocyte‐derived EVs from human primary astrocytes: (a) Quantification of HPA‐derived EVs (ADEVs) by ZetaView. (b) Representative western blot images showing expression of EV markers (Alix, TSG 101, CD 63, Flotiliin and Ago2). (c) Representative Transmission Electron Microscopy (TEM) Images of different fractions of EVs (Scale bar: 100 nm). (d) Representative western blot (WB) images showing protein levels of APP and AβmOC64 in different fractions of ADEVs. (e) Representative WB images showing protein levels of APP and AβmOC64 in 167 × 1000 g fractions of ADEVs at different time points. Data are presented as mean ± SEM. One‐way ANOVA followed by Bonferroni post hoc test was performed, *p < 0.05 versus control. n = 6 biological replicates (HPAs from six‐donors)/group. ADEVs, astrocyte derived‐extracellular vesicles; Aβ‐ amyloid beta; Ago, Argonuate; ALIX, ALG‐2 interacting protein X; APP, amyloid precursor protein; CD63, cluster of differentiation; HT, heated Tat; Tat, transactivator of transcription; TSG101, tumour susceptibility gene 101.

Fig 4: ADEVs in dissemination of Alzheimer's like pathology: (a)–(b) Representative WB images of the Aβ and pTau after ADEV exposure from different groups. (c) Representative IHC images of the Aβ and pTau after ADEV exposure from different groups. (d) Electrophysiological analysis—mEPSCs in ADEV exposed neurons. Data are presented as mean ± SEM. One‐way ANOVA followed by Bonferroni post hoc test was performed, *p < 0.05 versus CEV/C, #p < 0.05 versus TEV. Scale bar: 10 μm. n = 6 (ADEVs from six‐donors on neurons isolated from the pups of six‐pregnant rats). CEV, ADEVs from scrambled siRNA transfected HPAs; C, control ADEV; GW, GW4869 (inhibitor of EV‐biogenesis) ADEVs; HCEV, ADEVs from HIF‐1α siRNA transfected HPAs, HT, Heated Tat ADEV; HTEV, ADEVs from HIF‐1α siRNA transfected Tat exposed HPAs; mEPSC, miniature excitatory post‐synaptic currents; MT, Mutant Tat ADEV; PSD95, post synaptic density protein 95; T, Tat‐ADEV; TEV, ADEVs from scrambled siRNA transfected Tat exposed HPAs; vGLUT1, vesicular glutamate transporter‐1.

Fig 5: Immunohistochemistry in ADEV injected mice: Representative IHC images of different brain regions demonstrating the expression of MAP2, Drebrin in the hippocampus (a), frontal cortex (b) and respective quantification (c)–(d). Representative IHC images of different brain regions demonstrating the expression of MAP2, Drebrin in the hippocampus (e), frontal cortex (f) and respective quantification (g)–(h). Data are presented as mean ± SEM. One‐way ANOVA followed by Bonferroni post hoc test was performed, *p < 0.05 versus CEV, #p < 0.05 versus TEV. n = 6 mice/group. Scale bar: 20 μm. CEV, ADEVs from scrambled siRNA transfected HPAs; GAD65, glutamic acid decarboxylase 65‐kilodalton isoform; GFAP, Glial‐fibrillary acidic protein; HCEV, ADEVs from HIF‐1α siRNA transfected HPAs, HTEV, ADEVs from HIF‐1α siRNA transfected Tat exposed HPAs; Iba1, ionized calcium‐binding adapter molecule 1; Syp, Synaptophysin; TEV, ADEVs from scrambled siRNA transfected Tat exposed HPAs.