Behavioral effects of SQSTM1/p62 overexpression in mice: support for a mitochondrial role in depression and anxiety

Abstract

Affective spectrum and anxiety disorders have come to be recognized as the most prevalently diagnosed psychiatric disorders. Among a suite of potential causes, changes in mitochondrial energy metabolism and function have been associated with such disorders. Thus, proteins that specifically change mitochondrial functionality could be identified as molecular targets for drugs related to treatment for affective spectrum disorders. Here, we report generation of transgenic mice overexpressing the scaffolding and mitophagy related protein Sequestosome1 (SQSTM1/p62) or a single point mutant (P392L) in the UBA domain of SQSTM1/p62. We show that overexpression of SQSTM1/p62 increases mitochondrial energy output and improves transcription factor import into the mitochondrial matrix. These elevated levels of mitochondrial functionality correlate directly with discernible improvements in mouse behaviors related to affective spectrum and anxiety disorders. We also describe how overexpression of SQSTM1/p62 improves spatial learning and long term memory formation in these transgenic mice. These results suggest that SQSTM1/p62 provides an attractive target for therapeutic agents potentially suitable for the treatment of anxiety and affective spectrum disorders.

Fig. 1

Demonstration that EFGP tagged proteins were effectively expressed in mouse brain. A) Overexpressed EGFP-p62 protein was mainly localized to the hippocampus in transgenic mice. Mouse brain from either OEp62 or p62:P392L expressing mice were dissected into regions as indicated (HYPO=hypothalamus; CRBLM=cerebellum; BRST=brain stem; HC=hippocampus; STRIA=striatum; CTX=cortex) and Western blotted with p62^Hum antibody to detect human specific overexpressing p62 protein. β-actin levels were used as control to indicate loading. B) Sagittal plane slices (200μm) were made through the hippocampal region of mouse brains from OEp62 and p62:P392L mice and examined under a fluorescent microscope at 488nm to show localization of EGFP tagged proteins in the hippocampal region. Bright field images are provided for structural reference. CA1, CA3 and DG regions in the hippocampus are labeled for orientation. Scale bar = 300μm.

Fig. 2

p62 overexpression increases the functionality of mitochondria in the hippocampus. A) Mitochondrial morphology was altered in OEp62 mice compared to WT and p62:P392L. Primary neuronal cells were cultured from dissected embryonic Day 19 hippocampus. At culture day 7, mitochondria were visualized by staining with MitoTracker Red and examined using a confocal microscope. B) Activated AMPK levels were decreased in p62 overexpressing tissue. Hippocampal lysates from WT, OEp62 and p62:P392L mice were separated on SDS-PAGE and phospho-AMPK levels compared to total AMPK examined by Western blot. C) Overexpression of p62 results in increased ATP production. Total ATP levels in hippocampal lysates were measured by luciferase assay and compared to WT. D) Mitochondrial import was increased with p62 overexpression. Mitochondria were isolated from hippocampal lysates and import of TFAM examined by SDS-PAGE and Western blot. Presence of overexpressed protein was confirmed with p62^Hum antibody and compared to native protein. E) p62 overexpression did not show increased mtDNA levels. Total mitochondrial DNA copy number was quantitated by RT-PCR.

Fig. 3

p62 overexpression influences affective disorder behavior patterns. A) Behavior in the Open Field Maze: there was no significant difference in distance traveled in the maze between genotypes. B) Open field behavior analyzed by zone: p62 overexpression resulted in increased maze exploration toward to the center and away from the walls by genotypes. C) Anxiety measure in the Elevated Plus Maze: overexpression of p62 substantially reduced the time spent in open arms of the maze, however, expression of P392L protein increased time spent on open arms. D) Depression measure in the Forced Swim Test: no difference in behavior was indicated when using immobility time as a measure. E) Mean behavior exhibited during the Forced Swim Test: overexpression of p62 changed the overall behavior patterns resulting in increased swimming activity and decrease climbing compared to WT. Refer to Table 1 for analysis of statistics.

Fig. 4

p62 overexpression affects spatial learning and memory tasks. A) Latency times to complete the Barnes Maze: overexpression of p62 improved latency times compared to WT on last day of Acquisition period. B) Probe Trials in the Barnes Maze (1=Probe Trial 2 hours post Day 5 acquisition; 2=Probe Trial 6 days post Day 5 acquisition): no significant differences in short term memory measure during Probe Trial 1 were observed between genotypes, however overexpression of p62 improved long term memory in Probe Trial 2 compared to WT and p62:P392L. Refer to Table 1 for analysis of statistics.