Localization and regulation of catabolic enzymes of the endocannabinoid pathway

distribution and regulation of ABHD6 and FAAH in greater detail. To this end the specificity of the antibodies was tested using shRNA mediated KD of both enzymes. Cells were transfected with the ABHD6 KD construct or a scrambled control at DIV 9. To check for transfection of the cells EGFP was coexpressed from the same plasmid. The cells were fixed at DIV 14 and immunostained for ABHD6 (Figure 16A). The ABHD6 signal was measured in the dendrites of transfected cells using the EGFP signal as a mask. The ABHD6 signal was significantly reduced following KD suggesting that the antibody is specific for the target sequence. Cells were transfected with the FAAH KD construct or a scrambled control at DIV 9. To control for transfection of the cells EGFP was coexpressed from the same plasmid. The cells were fixed at DIV 14 and immunostained for ABHD6 (Figure 16A). The FAAH signal was measured in the dendrites of transfected cells using the EGFP signal as a mask. The FAAH signal was significantly reduced following KD suggesting that the antibody is specific for the target. Since both ABDH6 and FAAH are involved in the degradation of CB1R agonists, it is plausible that they might localize in close proximity to CB1R-containing presynapses. Presynaptic boutons were labeled using the synaptic vesicle protein Synaptophysin (SYP). To label dendrites and spines postsynaptic cells were filled with EGFP as a volume marker and stained for ABHD6 or FAAH (Figure 16E, F). The percentage of spines contacting a SYP-labeled presynapse containing ABHD6 and the percentage of those spines contacting a CB1R-containing presynapse was counted. 40% of all spines contained ABHD6; of those spines 80% contacted a CB1R positive bouton (Figure 16F). The percentage of spines contacting a SYP-labeled presynapse containing FAAH and the percentage of those spines contacting a CB1R containing presynapse was counted. 40% of all spines contained ABHD6; of those spines 80% contacted a CB1R positive bouton (Figure 16H).

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Figure 16. ABHD6 and FAAH localization and KD verification

A-B: ABHD6 intensities were measured in cells transfected with ABHD6 KD (n=16) or scrambled control (n=17). Scale bar: 5 µm

C-D: FAAH intensities were measured in cells transfected with FAAH KD (n=8) or scrambled control (n=8). Scale bar: 5 µm

E-H: Hippocampal primary neurons were transfected at DIV 20 with GFP-N1 and fixed after 24 h at DIV 21. The cells were stained for CB1R, SYP and ABHD6 (n=8) (E). Total localization of ABHD6 with the synaptic marker SYP and with synapses labeled by both SYP and CB1R were analyzed (F). GFP transfected cells were stained for CB1R, SYP and FAAH (n=10) (G).

Total localization of FAAH with the synaptic marker SYP and with synapses labeled by both SYP and CB1R were analyzed (H). Scale bar: 5 µm

Data information: Data are presented as boxplots whiskers minimum to maximum.

**** p≤0.0001 (Student's t-test).

We then studied how the synaptic levels of FAAH and ABHD6 are regulated using primary hippocampal neurons. To analyze the levels of both proteins, synapses were immunolabeled

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for the excitatory synapse marker Shank3 (Figure 17A, C, E, G). Since EE results in an increase in synaptic activity (Ohline and Abraham, 2019b), the effect of synaptic silencing on the synaptic localization of FAAH and ABHD6 was studied, by bath application of TTX in the culture medium for 48 h (Figure 17A-D). Synaptic ABHD6 levels were not affected by synaptic silencing (Figure 17B); the synaptic expression of FAAH on the other hand was reduced following synaptic silencing (Figure 17D). It was previously shown that a positive feedback loop exists between presynaptic CB1R activation and postsynaptic 2-AG synthesis (Anderson et al., 2015). To explore the effect of CB1R signaling on the synaptic localization of ABHD6 and FAAH, the cells were treated with the CB1R-antagonist AM-251 (Figure 17E-H). Inhibition of CB1R signaling resulted in a reduction of synaptic ABHD6 levels (Figure 17F), but did not affect the synaptic levels of FAAH (Figure 17G). This suggests that synaptic localization of ABHD6 is dependent on synaptic 2-AG production.

Figure 17. Regulation of ABHD6 and FAAH levels in hippocampal primary neurons

A-D: Hippocampal neurons were silenced by 1 μM TTX for 48 h (A, D). The cells were stained for MAP2, Shank3 and ABHD6 (n=28) (G) or FAAH (n=9) (H) at DIV 21. The levels of ABHD6 (G) and FAAH (H) were measured in Shank3 labeled synapses.

E-H: Hippocampal neurons were treated for 48 h with the CB1R antagonist AM-251 for 48 h (E, G).

The cells were stained for MAP2, Shank3 and ABHD6 (n=18) (F) or FAAH (n=23) (H) at DIV 21. The levels of ABHD6 (F) and FAAH (H) were measured in Shank3 labeled synapses.

Data information: Data are presented as boxplots whiskers minimum to maximum.

*** p≤0.001 (Student's t-test).

We then aimed to get a more detailed understanding of the subcellular localization of ABHD6. Mature hippocampal rat neurons (DIV 21) were transfected with a MARCKS-GFP

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expressing construct, labeling the cell membrane. The cells were stained with antibodies against ABHD6 and Shank3 (Figure 18A). Imaging at STED-resolution revealed clusters of ABHD6 associated with the postsynaptic density (PSD), in a subset of spines (Figure 18A 1, 2), with some spines lacking ABHD6 (Figure 18A, 3). ABHD6 is known as both a postsynaptic and an ER protein; it was therefore tested if ABHD6 is localized to spines containing a type of ER membrane known as the spine apparatus. Mature hippocampal rat neurons were stained for the spine apparatus marker Synaptopodin, Shank3 and ABHD6. 20%

of Shank3 labeled synapses contained Synaptopodin, of these synapses 80% also contained ABHD6, while only 36% of Shank3 labeled synapses contained ABHD6 (Figure 18C, D).

This suggests that the presence of the spine apparatus is not necessary for the localization of ABHD6 in spines, but that spines containing a spine apparatus are highly likely to contain ABHD6.

Figure 18. Synaptic distribution of ABHD6

A: Cells were transfected with MARCKS-GFP on DIV 15 and fixed on DIV 16. Cells were stained against Shank3 and ABHD6. Individual spines with ABHD6 (1, 2) and without ABHD6 (3) are shown.

B: Cells DIV 16 were stained for ABHD6, Synaptopodin and Shank3 (n=15).

C-D: Colocalization of ABHD6 and Synaptopodin with Shank3 and the colocalization of Synaptopodin and Shank3 positive synapses with ABHD6.

Data information: Data are presented as boxplots whiskers minimum to maximum.

ABHD6 is an ER-resident enzyme and most interestingly its reduced postsynaptic localization following blockage of CB1R came along with a displacement of the Synaptopodin-positive spine apparatus (Figure 19). To analyze if the regulation of synaptic ABHD6 levels might be dependent on the spine apparatus, hippocampal primary neurons were again treated with AM-251 for 48 h. corresponding to the effect on synaptic ABHD6 the number of spines containing

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Synaptopodin was also reduced following inhibition of CB1R signaling (Figure 19). Thus, most likely a retraction of ER membranes from spines of CB1R bearing synapses underlies the removal of ABHD6 from synapses in response to reduced ECS.

Figure 19. Inhibition of CB1R decreases the number of spine apparatus containing synapses A, B: Cells were treated with AM-251 for 48 h and stained for Shank3 and Synaptopodin (SynPo)

(A) (n=11), Scale bar: 5 µm. Co-staining of Synaptopodin containing spines after blockage of CB1R with AM- 251 reveals a displacement of the Synaptopodin-positive spine apparatus (B).

Data information: Data are presented as boxplots with whiskers from minimum to maximum value. **p≤0.01 (Student's t-test).

The major 2-AG degrading enzyme is Monoacylglycerol lipase (MAGL). Several studies point to a predominant role of MAGL in the catabolism of 2-AG also at hippocampal synapses (Horváth et al., 2014; Guggenhuber et al., 2016; Wang et al., 2017). However, in contrast to FAAH and ABHD6 the levels of MAGL were not changed in mice subjected to EE in the proteomic analysis (Figure 10). Importantly, MAGL is a membrane-associated, but not a transmembrane protein and is thought to predominantly localize to presynapses (Hashimotodani et al., 2007). Further analysis of the mechanisms of altered ECS following EE therefore required substantiating these findings in primary neurons. Hippocampal primary neurons (DIV 21) were stained for MAGL, CB1R and the presynaptic marker SYP. MAGL indeed localized mainly to CB1R positive presynapses (Figure 20A), but not to CB1R negative synapses or extrasynaptic CB1Rs. To further prove that MAGL is indeed specifically localized to presynapses and not to dendrites and spines, hippocampal neurons were transfected with GFP as a cell fill and stained for MAGL (Figure 20B). It turned out that MAGL is predominantly localized outside of dendrites, excluding a major function in control of postsynaptic 2-AG catabolism.

56 Figure 20. Synaptic localization of MAGL

A: Hippocampal primary neurons stained for CB1R (blue), MAGL (magenta) and SYP (green) at DIV21

B: Hippocampal neurons filled with GFP stained for MAGL at DIV 21