TRIAD3/RNF216 mutations associated with Gordon Holmes syndrome lead to synaptic and cognitive impairments via Arc misregulation

Summary Multiple loss‐of‐function mutations in TRIAD3 (a.k.a. RNF216) have recently been identified in patients suffering from Gordon Holmes syndrome (GHS), characterized by cognitive decline, dementia, and movement disorders. TRIAD3A is an E3 ubiquitin ligase that recognizes and facilitates the ubiquitination of its target for degradation by the ubiquitin‐proteasome system (UPS). Here, we demonstrate that two of these missense substitutions in TRIAD3 (R660C and R694C) could not regulate the degradation of their neuronal target, activity‐regulated cytoskeletal‐associated protein (Arc/Arg 3.1), whose expression is critical for synaptic plasticity and memory. The synaptic deficits due to the loss of endogenous TRIAD3A could not be rescued by TRIAD3A harboring GHS‐associated missense mutations. Moreover, we demonstrate that the loss of endogenous TRIAD3A in the mouse hippocampal CA1 region led to deficits in spatial learning and memory. Finally, we show that these missense mutations abolished the interaction of TRIAD3A with Arc, disrupting Arc ubiquitination, and consequently Arc degradation. Our current findings of Arc misregulation by TRIAD3A variants suggest that loss‐of‐function mutations in TRIAD3A may contribute to dementia observed in patients with GHS driven by dysfunctional UPS components, leading to cognitive impairments through the synaptic protein Arc.

Imaging was performed in Tyrode's solution (139 mM NaCl, 3 mM KCl, 17 mM NaHCO 3 , 12 mM glucose, 3 mM CaCl 2 , and 1 mM MgCl 2 ), and cells were maintained at 37°C during the experiments. The TIRFM setup consisted of a Nikon Eclipse Ti inverted microscope (Nikon) equipped with an EMCCD camera (Andor Technology), 100x oil immersion objective (1.45 NA) and argon (488 nm) and argon/krypton (568 nm) lasers. Focal drift was prevented by an in-built autofocus assist system.
All data were auto-sorted followed by manually removing false events with wrong trace profile. The rise and decay kinetics were comparable across analysed groups.

In vivo ubiquitination assay
The in vivo ubiquitination assay was performed as described previously (Mabb et al. 2014). Briefly, HEK293T cells transfected with the FLAG-TRIAD3A-WT, FLAG-TRIAD3A-R660C, or FLAG-TRIAD3A-R660C constructs were transfected into HEK293T cells along with Myc-Arc and HA-ubiquitin. The cell lysates were prepared the next day using IP buffer containing 1% SDS and subsequently boiled for 20 minutes. The cell debris was removed by centrifugation, and the samples were diluted 10 times with IP buffer containing phosphatase and protease inhibitors, 10 mM N-ethylmaleimide, and 3 mM iodoacetamide so that the final SDS concentration was 0.1%. Arc was immunoprecipitated using anti-Myc beads (Sigma #A7470), washed 3 times, and subjected to western blot following gel electrophoresis. The membrane was probed with anti-HA antibody (Covance #MMS-101R) to detect Arc ubiquitination.

Behavioral procedures
Open field (OF) test: For OF experiments, the animals were placed in the center of an acrylic animal cage for 1 hour, and their activity was assessed and calculated every 5 minutes using a VersaMax activity monitoring system. was placed in the west arm of the water-filled maze, and 6 trials were performed each day for 4 days for the animal to learn the location of the platform. Each trial was conducted after blocking the arm opposite to the start arm (S or N), effectively mimicking a T-maze. Among the 6 trials carried out each day, 3 trials were started from the south arm, and the remaining three were initiated from the north arm in a randomized order. During each trial, the animal was given 30 seconds to locate the platform, after which it was gently prodded to the platform. The time taken to reach the platform viz latency, the direct navigation from the start arm (S or N) to the arm containing the platform (W) viz accuracy, and the number of visits to half the distance of the arm opposite arm without the platform (E) viz wrong platform visits, are considered measures of learning. One or two-way analysis of variance (ANOVA) was used according to the number of factors considered followed by Sidak's post hoc comparisons where applicable.
Morris water maze (MWM): The water maze consisted of a 120 cm diameter grey circular pool filled with water (23-26 • C, 40 cm deep) made opaque by adding non-toxic white paint (Crayola). The pool was surrounded by several distant cues from the environment of the experimental room. The animals learned to find a transparent platform (10 cm in diameter) hidden 1 cm below the water surface; the location of the platform remained constant throughout the experiment. Each animal was tested four times a day across five consecutive days, with an intertrial of approximately 45 minutes. For each trial, the mice were released facing the tank wall from one randomly selected starting points (N, S, E or W) and were allowed to swim until they reached the platform. Animals that failed to find the platform within 1 minute (60 seconds) were gently directed to it and put on it for 15 seconds. After the trial, the mice were removed from the pool, gently dried with a towel, put individually in cages filled with paper towel, and warmed with water bottles to avoid hypothermia. The criterion of learning success consisted of reaching the platform in less than 20 seconds. On the 6th day, a 60-second probe trial was conducted without platform and the time spent in each quadrant was analyzed. Trials were recorded with a video camera placed above the center of the pool, and the analysis was automated through the ANY-maze video tracking system (Stoelting, USA). Data (means and standard error of the means) were analyzed with IBM SPSS Statistics v19.0 software. One or two-way ANOVA was used according to the number of factors considered ("treatment", time in "days" or "trials", and "quadrants"), followed by Bonferroni post hoc comparisons if needed. Mice that remained immobile for more than 25 seconds in 7 or more trials during the training phase were excluded from the analyses.   (n=8)).

Figure 6C
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