The retrotransposon - derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

The human genome contains 24 gag -like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag -like genes PNMA1 and PNMA4 support reproductive capacity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. Analysis of donated human ovaries shows that expression of both genes declines normally with aging, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

Dosage sensitivity to Pumilio1 variants in the mouse brain reflects distinct molecular mechanisms.

Different mutations in the RNA-binding protein Pumilio1 (PUM1) cause divergent phenotypes whose severity tracks with dosage: a mutation that reduces PUM1 levels by 25% causes late-onset ataxia, whereas haploinsufficiency causes developmental delay and seizures. Yet PUM1 targets are derepressed to equal degrees in both cases, and the more severe mutation does not hinder PUM1's RNA-binding ability. We therefore considered the possibility that the severe mutation might disrupt PUM1 interactions, and identified PUM1 interactors in the murine brain. We find that mild PUM1 loss derepresses PUM1-specific targets, but the severe mutation disrupts interactions with several RNA-binding proteins and the regulation of their targets. In patient-derived cell lines, restoring PUM1 levels restores these interactors and their targets to normal levels. Our results demonstrate that dosage sensitivity does not always signify a linear relationship with protein abundance but can involve distinct mechanisms. We propose that to understand the functions of RNA-binding proteins in a physiological context will require studying their interactions as well as their targets.

Dual antibody strategy for high-resolution imaging of murine Purkinje cells and their dendrites across multiple layers.

Despite being among the largest neurons in the mammalian brain, Purkinje cells are difficult to visualize and trace via immunofluorescence because their dendritic arbors extend through several cerebellar layers. This protocol describes a two-antibody strategy we developed to study Purkinje cell morphology in mice. With it, one can reconstruct three-dimensional images of Purkinje cells at single-neuron resolution across multiple layers. The substantially improved image quality reveals subtle defects, enabling more meaningful morphological analysis. For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2015).

Protocol to assess the effect of disease-driving variants on mouse brain morphology and primary hippocampal neurons.

Genetic variants that affect neurological function will often produce changes visible at the level of gross morphology, either of the whole brain or of specific neuronal types. Here we describe how to perfuse and dissect the brain in preparation for Nissl staining. Then we outline steps for culturing mouse primary hippocampal neurons to evaluate dendritic arborization (Sholl analysis). For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2018).

Determining the effects of loss of function mutations in human cell lines.

Quantifying differences in the amount of protein and mRNA caused by missense mutations in a gene of interest can be challenging, especially when using patient-derived primary cells, which are intrinsically variable. In this protocol, we describe how to culture patient-derived lymphoblast and fibroblast cell lines for later mRNA and protein quantification. We also describe the steps to examine variants of PUM1 in HEK293T cells, but the protocol can be applied to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2018).

5-HT2C receptor blockade reverses SSRI-associated basal ganglia dysfunction and potentiates therapeutic efficacy.

Serotonin (5-HT) selective reuptake inhibitors (SSRIs) are widely used in the treatment of depression and anxiety disorders, but responsiveness is uncertain and side effects often lead to discontinuation. Side effect profiles suggest that SSRIs reduce dopaminergic (DAergic) activity, but specific mechanistic insight is missing. Here we show in mice that SSRIs impair motor function by acting on 5-HT2C receptors in the substantia nigra pars reticulata (SNr), which in turn inhibits nigra pars compacta (SNc) DAergic neurons. SSRI-induced motor deficits can be reversed by systemic or SNr-localized 5-HT2C receptor antagonism. SSRIs induce SNr hyperactivity and SNc hypoactivity that can also be reversed by systemic 5-HT2C receptor antagonism. Optogenetic inhibition of SNc DAergic neurons mimics the motor deficits due to chronic SSRI treatment, whereas local SNr 5-HT2C receptor antagonism or optogenetic activation of SNc DAergic neurons reverse SSRI-induced motor deficits. Lastly, we find that 5-HT2C receptor antagonism potentiates the antidepressant and anxiolytic effects of SSRIs. Together our findings demonstrate opposing roles for 5-HT2C receptors in the effects of SSRIs on motor function and affective behavior, highlighting the potential benefits of 5-HT2C receptor antagonists for both reduction of motor side effects of SSRIs and augmentation of therapeutic antidepressant and anxiolytic effects.

Activity of Raphé Serotonergic Neurons Controls Emotional Behaviors.

Despite the well-established role of serotonin signaling in mood regulation, causal relationships between serotonergic neuronal activity and behavior remain poorly understood. Using a pharmacogenetic approach, we find that selectively increasing serotonergic neuronal activity in wild-type mice is anxiogenic and reduces floating in the forced-swim test, whereas inhibition has no effect on the same measures. In a developmental mouse model of altered emotional behavior, increased anxiety and depression-like behaviors correlate with reduced dorsal raphé and increased median raphé serotonergic activity. These mice display blunted responses to serotonergic stimulation and behavioral rescues through serotonergic inhibition. Furthermore, we identify opposing consequences of dorsal versus median raphé serotonergic neuron inhibition on floating behavior, together suggesting that median raphé hyperactivity increases anxiety, whereas a low dorsal/median raphé serotonergic activity ratio increases depression-like behavior. Thus, we find a critical role of serotonergic neuronal activity in emotional regulation and uncover opposing roles of median and dorsal raphé function.

Postnatal day 2 to 11 constitutes a 5-HT-sensitive period impacting adult mPFC function.

Early-life serotonin [5-hydroxytryptamine (5-HT)] signaling modulates brain development, which impacts adult behavior, but 5-HT-sensitive periods, neural substrates, and behavioral consequences remain poorly understood. Here we identify the period ranging from postnatal day 2 (P2) to P11 as 5-HT sensitive, with 5-HT transporter (5-HTT) blockade increasing anxiety- and depression-like behavior, and impairing fear extinction learning and memory in adult mice. Concomitantly, P2-P11 5-HTT blockade causes dendritic hypotrophy and reduced excitability of infralimbic (IL) cortex pyramidal neurons that normally promote fear extinction. By contrast, the neighboring prelimbic (PL) pyramidal neurons, which normally inhibit fear extinction, become more excitable. Excitotoxic IL but not PL lesions in adult control mice reproduce the anxiety-related phenotypes. These findings suggest that increased 5-HT signaling during P2-P11 alters adult mPFC function to increase anxiety and impair fear extinction, and imply a differential role for IL and PL neurons in regulating affective behaviors. Together, our results support a developmental mechanism for the etiology and pathophysiology of affective disorders and fear-related behaviors.