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. 2022 Jun 22;29:189–203. doi: 10.1016/j.omtn.2022.06.015

Figure 5.

Figure 5

TECPR2 and TECPR2ΔExon8 neuronal localization in patient iPSC-derived neurons

(A–D′) High-resolution immunocytochemistry analyses of control TECPR2+/+ iPSC neurons (DIV45) treated with 5 μM scrambled non-targeting negative control ASO (scASO), single dose, gymnotic delivery at plating. Neurons were co-stained for MAP2 (A), β-III TUBULIN (B), and TECPR2 (D). β-III TUBULIN/TECPR2 merged signal (C), and an enlarged (∼2.1×) sub-region of the TECPR2 image (D′) are also presented. Distinct patterns of TECPR2 sub-cellular localization are highlighted in (D) and (D′): broad, speckled puncta pattern within soma and proximal dendrites (cyan arrowhead), small discrete puncta in neurites (yellow arrowhead), and rare large puncta in neurites (green arrowheads). (E–H′) Control TECPR2+/- iPSC neurons (DIV45) treated with 5 μM scASO, single dose, gymnotic delivery show similar pattern of TECPR2 subcellular localization to that of TECPR2+/+ (D and D′) with the three types of puncta highlighted by arrowheads in (H) and (H′): broad puncta pattern within soma and proximal dendrites (cyan arrowhead), small discrete puncta in neurites (yellow arrowhead), and rare large puncta in neurites (green arrowheads). (I–L′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with the 5 μM scASO show no TECPR2 immunoreactivity and absence of the punctate expression pattern. White arrowhead in (L) and (L′) indicates lack of TECPR2 immunoreactivity signal in an area of a MAP2+ soma, according to (I). (M–P′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of broad puncta pattern of TECPR2 expression within soma and proximal dendrites, as highlighted by cyan arrowhead in (P) and enlarged (∼2.1×) in (P′). (Q–T′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of expression of small discrete TECPR2-positive puncta in β-III TUBULIN-positive neurites, as highlighted by yellow arrowheads in (T) and enlarged (∼2.1×) (T′). (U–X′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of expression of rare large TECPR2-positive puncta in neurites, as highlighted by green arrowhead in (X) and enlarged (∼2.1×) (X′). (A–X′) Scale bar, 20 μm.