Mutant screen for reproduction unveils depression-associated Piccolo’s control over reproductive behavior

Abstract

Successful sexual reproduction involves complex, genetically encoded interplay between animal physiology and behavior. The rat provides a highly fecund mammalian model for studying how the brain impacts reproduction. Here, we report a forward genetics screen in rats to identify genes that affect reproduction. A panel of 18 distinct rat strains harboring Sleeping Beauty gene trap mutations were analyzed for the ability to reproduce. As expected, our mutant screen identified genes where reproductive failure was connected to gametogenesis (Btrc, Pan3, Spaca6, Ube2k) and embryogenesis (Alk3, Exoc6b, Slc1a3, Tmx4, Zmynd8). In addition, we identified Atg13 (longevity) and Pclo (neuronal disorders), previously not associated with an inability to conceive.Neurologically, Pclo is known to regulate the size of presynaptic vesicle pools. Here, dominant traits in Pclo mutant rats caused epileptiform activity and affected genes supporting GABAergic synaptic transmission (Gabra6, Gabrg3). Recessive traits in Pclo mutant rats transmitted altered reproductive behavior, as homozygous Pclo mutant rats produced gametes but neither sex would mate with wildtype rats. Pclo mutant rat behavior was linked to endophenotypes signifying compromised brain-gonad crosstalk via disturbed GnRH signaling and allelic markers for major depressive disorder in humans (Grm5, Htr2a, Sorcs3, Negr1, Drd2). Thus, by rat genetics, we identified Pclo as a candidate presynaptic factor required for reproduction. Author SummaryPiccolo gene mutations have previously been identified in human cohorts diagnosed with behavioral syndromes that impact one's emotions, including depression and bipolar disorder.Although studies in human populations implicate changes to Piccolo's DNA sequence to enhanced susceptibility for behavioral disorders, studies in mouse models have yet to link Piccolo mutations to altered behavior. Here, by a novel genetics approach, we report Piccolo mutationdependent effects on reproductive behavior in rats, a finding that may turn out to be relevant to the behavioral effects that are associated with human Piccolo gene mutations. Thus, research aimed at understanding how Piccolo functions to regulate reproduction in rats could prove pivotal in our ability to understand neurological mechanisms that influence human emotions.pathologies, and in males, Atg13 gt/gt was further complicated with abnormal spermatozoa. Thus, the cause of infertility of Atg13 gt/gt rats is a cumulation of general fitness problems. Compromised neurotransmission in Pclo mutantsDespite their reproductive failure (Fig 2A), Pclo gt/gt mutant rats did not display any obvious dysfunction during gametogenesis (S2A and S2C Fig). Numbers of epididymal spermatozoa from Pclo gt/gt rats were relatively normal (Fig 2C). However, spermatozoa from Pclo gt/gt rats were not found in vaginal swabs of WT females (6 of 6 breeder pairs) (Fig 4A, and spermatozoa from WT males were not detected in Pclo gt/gt females (6 of 6 breeder pairs) (Fig 4A), suggesting that the mating did not actuall...

Loss of Piccolo function in rats induces Pontocerebellar Hypoplasia type 3-like phenotypes

“…Intriguingly, Pclo gt/gt rats display a reduced expression of the GABA A α6 subunit at MF rosettes (Figure 6). This is in line with findings from Medrano et al (2019), who performed RNA sequencing on the Pclo gt/gt rats and found a severe reduction in GABA A α6 subunit gene expression.…”

“…In behavioral tests, we observed that Pclo gt/gt rats performed significantly worse than both Pclo wt/wt and Pclo wt/gt littermates at motor function tasks, highlighting the recessive nature of the behavioral impairments. Our colleagues Medrano et al (2019) also observed epileptic seizures and increased aggression in Pclo gt/gt rats, and a failure to reproduce due to impaired brain-gonad signaling.…”

“…In addition to brain atrophy, children with PCH3 have, amongst other symptoms, cognitive and motor deficits as well as seizures. Given that seizures were also observed in Pclo gt/gt rats (Medrano et al, 2019), we postulate that Pclo gt/gt rats represent a model to study the underlying mechanisms of this devastating disease.…”

“…It is thus possible that, in the absence of Piccolo, Dvl1 is not properly localized to presynaptic sites, preventing proper Wnt signaling and consequently causing the formation of smaller and less organized MF-GC synapses. Furthermore, analysis of Pclo gt/gt transcripts reveals that Wnt expression is reduced (Medrano et al, 2019). Thus, there appears to be at least two possible mechanisms that could contribute to smaller MF boutons in Piccolo knockout rats: defects in SV recycling and Wnt signaling.…”

“…What remains unclear is how Piccolo contributes to cerebellar development and whether, as suggested by genetic studies, it has a primary role in the etiology of PCH3. The recent generation of a Piccolo knockout rat ( Pclo gt/gt ) using transposon mutagenesis (Medrano et al, 2019) provides an opportunity to explore this potential relationship.…”