Fig 2: Histological analysis of brain tissues using H&E staining. Brain sections from healthy (A) or CLN2 mice (10 d. old) treated with saline (B), EV-TPP1 (C), or sham EVs (D), via combination of i.p. and i.t. injections as described in the Materials and Methods section. Normal Purkinje cells displaced in the molecular layer (M) (arrow) and cell dendrites (arrows) detected mostly in brain tissues recovered from healthy control (A) and EV-TPP1-treated CLN2 (C) mice. Densely populated granule cell layer (G) and the uniform molecular layer (M) in the healthy control (A) and EV-TPP1-treated CLN2 (C) brain section. Hypocellular granule cell layer (G), an absence of Purkinje cells, and a vacuolated and loosely arranged molecular layer (M) in saline-treated CLN2 (B) and sham-EV-treated CLN2 (D) brain sections.

Fig 3: Effect of EV-TPP1 treatment on expression of P62 in brain of CLN2 mice. CLN2 mice (10 d. old) were treated with saline, EV-TPP1, sham EVs, or TPP1 alone via combination of i.p. and i.t. injections as described in the Materials and Methods section. Wild type control mice were injected with saline. Three months later, animals were sacrificed, and sliced brain tissues were stained with antibody against SQSTM1/P62 (A). Nuclei were visualized with DAPI staining. SQSTM1/P62 immunoreactivity was measured using Zen software, and the numbers of cells expressing the protein were plotted in box and whiskers plots using GraphPad Prism (B). Relative mRNA expression of the indicated genes was measured in brain tissues using autophagy PCR arrays, and the numbers are plotted in a heat map as fold change from CLN2/saline (left panel) and as fold change from WT/saline (right panel) using GraphPad Prism (C). Data were analyzed using one- or two-way ANOVA analysis followed by Tukey’s multiple comparisons test. *** p < 0.0001; ** p < 0.005; * p < 0.05. Scale bar: 20 µm.

Fig 4: EV-TPP1 treatment resulted in significant elimination of storage accumulation (subunit C of mitochondrial ATP synthase) in the brains of CLN2 mice. CLN2 mice (10 d. old) were treated with saline, EV-TPP1, sham EVs, or TPP1 alone via combination of i.p. and i.t. injections once a week. Wild type control mice were injected with saline. Three months later, animals were sacrificed, and sliced brain tissues were stained with antibody to subunit C of mitochondrial ATP synthase (A) as described in the Materials and Methods section. Immunohistochemical analysis of subunit C of mitochondrial ATP synthase (SCMAS) revealed a robust treatment effect of EV-TPP1 upon the treatment. This was not seen in sham-EVs-treated CLN2 mice. Scale bar: 50 µm.

Fig 5: Genetic modification of parent macrophages with TPP1-encoding pDNA and TPP1 levels in EVs released by the cells. (A) Macrophages were transfected with TPP1 pDNA tagged with Myc using the Invitrogen Neon Electroporation system with two different electroporation conditions. (B) Twenty-four h after the transfection, the expression of the encoded protein Myc-TPP1 was visualized with FITC anti-Myc tag antibody (ab1263, green). Nuclei were stained with DAPI (blue). (C,D) The kinetics of TPP1 expression in parent macrophages and EVs released by the cells were assessed by ELISA over four days after the transfection with condition #1 (black bars) and condition #2 (white bars). Scale bar: 20 µm. Data were analyzed using two-way ANOVA analysis. Values are means ± SEM.