Bryan W. Luikart

Pten Regulates Neuronal Arborization and Social Interaction in Mice

CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3beta. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.

TrkB Regulates Hippocampal Neurogenesis and Governs Sensitivity to Antidepressive Treatment

Adult hippocampal neurogenesis is stimulated by chronic administration of antidepressants (ADs) and by voluntary exercise. Neural progenitor cells (NPCs) in the dentate gyrus (DG) that are capable of continuous proliferation and neuronal differentiation are the source of such structural plasticity. Here we report that mice lacking the receptor tyrosine kinase TrkB in hippocampal NPCs have impaired proliferation and neurogenesis. When exposed to chronic ADs or wheel-running, no increase in proliferation or neurogenesis is observed. Ablation of TrkB also renders these mice behaviorally insensitive to antidepressive treatment in depression- and anxiety-like paradigms. In contrast, mice lacking TrkB only in differentiated DG neurons display typical neurogenesis and respond normally to chronic ADs. Thus, our data establish an essential cell-autonomous role for TrkB in regulating hippocampal neurogenesis and behavioral sensitivity to antidepressive treatments, and support the notion that impairment of the neurogenic niche is an etiological factor for refractory responses to an antidepressive regimen.

Brain-Derived Neurotrophic Factor Conditional Knockouts Show Gender Differences in Depression-Related Behaviors

BACKGROUND Indirect evidence suggests that loss of brain-derived neurotrophic factor (BDNF) from forebrain regions contributes to an individuals vulnerability for depression, whereas upregulation of BDNF in these regions is suggested to mediate the therapeutic effect of antidepressants. METHODS We have tested this hypothesis by generating two independent lines of conditional BDNF knockout mice in which the BDNF gene is deleted selectively in forebrain. RESULTS We show that male conditional knockouts exhibit hyperactivity but normal depression-related behaviors. In contrast, female conditional knockouts display normal locomotor activity but a striking increase in depression-like behavior. We also demonstrate that loss of BDNF in both male and female mice attenuates the actions of the antidepressant desipramine in the forced swim test. CONCLUSIONS These gender differences in depression-related behaviors in BDNF conditional knockout mice provide direct evidence for a role of BDNF in depression. The results also support the view that forebrain BDNF may be essential in mediating antidepressant efficacy.

microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus

Newborn neurons in the dentate gyrus of the adult hippocampus rely upon cAMP response element binding protein (CREB) signaling for their differentiation into mature granule cells and their integration into the dentate network. Among its many targets, the transcription factor CREB activates expression of a gene locus that produces two microRNAs, miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activity-dependent dendritic growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, miR-132 and -212 and their reverse strands miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling.

Conditional deletion of TrkB but not BDNF prevents epileptogenesis in the kindling model.

Epileptogenesis is the process whereby a normal brain becomes epileptic. We hypothesized that the neurotrophin brain-derived neurotrophic factor (BDNF) activates its receptor, TrkB, in the hippocampus during epileptogenesis and that BDNF-mediated activation of TrkB is required for epileptogenesis. We tested these hypotheses in Synapsin-Cre conditional BDNF(-/-) and TrkB(-/-) mice using the kindling model. Despite marked reductions of BDNF expression, only a modest impairment of epileptogenesis and increased hippocampal TrkB activation were detected in BDNF(-/-) mice. In contrast, reductions of electrophysiological measures and no behavioral evidence of epileptogenesis were detected in TrkB(-/-) mice. Importantly, TrkB(-/-) mice exhibited behavioral endpoints of epileptogenesis, tonic-clonic seizures. Whereas TrkB can be activated, and epileptogenesis develops in BDNF(-/-) mice, the plasticity of epileptogenesis is eliminated in TrkB(-/-) mice. Its requirement for epileptogenesis in kindling implicates TrkB and downstream signaling pathways as attractive molecular targets for drugs for preventing epilepsy.

TrkB Has a Cell-Autonomous Role in the Establishment of Hippocampal Schaffer Collateral Synapses

Neurotrophin signaling has been implicated in the processes of synapse formation and plasticity. To gain additional insight into the mechanism of BDNF and TrkB influence on synapse formation and synaptic plasticity, we generated a conditional knock-out for TrkB using the cre/loxp system. Using three different cre-expressing transgenic mice, three unique spatial and temporal configurations of TrkB deletion were obtained with regard to the hippocampal Schaffer collateral synapse. We compare synapse formation in mutants in which TrkB is ablated either in presynaptic or in both presynaptic and postsynaptic cells at early developmental or postdevelopmental time points. Our results indicate a requirement for TrkB at both the presynaptic and postsynaptic sites during development. In the absence of TrkB, synapse numbers were significantly reduced. In vivo ablation of TrkB after synapse formation did not affect synapse numbers. In primary hippocampal cultures, deletion of TrkB in only the postsynaptic cell, before synapse formation, also resulted in deficits of synapse formation. We conclude that TrkB signaling has a cell-autonomous role required for normal development of both presynaptic and postsynaptic components of the Schaffer collateral synapse.

Pten Knockdown In Vivo Increases Excitatory Drive onto Dentate Granule Cells

Some cases of autism spectrum disorder have mutations in the lipid phosphatase, phosphatase and tensin homolog on chromosome 10 (Pten). Tissue specific deletion of Pten in the hippocampus and cortex of mice causes anatomical and behavioral abnormalities similar to human autism. However, the impact of reductions in Pten on synaptic and circuit function remains unexplored. We used in vivo stereotaxic injections of lentivirus expressing a short hairpin RNA to knock down Pten in mouse neonatal and young adult dentate granule cells. We then assessed the morphology and synaptic physiology between 2 weeks and 4 months later. Confocal imaging of the hippocampus revealed a marked increase in granule cell size and an increase in dendritic spine density. The onset of morphological changes occurred earlier in neonatal mice than in young adults. We used whole-cell recordings from granule cells in acute slices to assess synaptic function after Pten knockdown. Consistent with the increase in dendritic spines, the frequency of excitatory miniature and spontaneous postsynaptic currents increased. However, there was little or no effect on IPSCs. Thus, Pten knockdown results in an imbalance between excitatory and inhibitory synaptic activity. Because reductions in Pten affected mature granule cells as well as developing granule cells, we suggest that the disruption of circuit function by Pten hypofunction may be ongoing well beyond early development.

miR-132 Mediates the Integration of Newborn Neurons into the Adult Dentate Gyrus

Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific ‘sponge’ containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.

Neurotrophin-Dependent Dendritic Filopodial Motility: A Convergence on PI3K Signaling

Synapse formation requires contact between dendrites and axons. Although this process is often viewed as axon mediated, dendritic filopodia may be actively involved in mediating synaptogenic contact. Although the signaling cues underlying dendritic filopodial motility are mostly unknown, brain-derived neurotrophic factor (BDNF) increases the density of dendritic filopodia and conditional deletion of tyrosine receptor kinase B (TrkB) reduces synapse number in vivo. Here, we report that TrkB associates with dendritic growth cones and filopodia, mediates filopodial motility, and does so via the phosphoinositide 3 kinase (PI3K) pathway. We used genetic and pharmacological manipulations of mouse hippocampal neurons to assess signaling downstream of TrkB. Conditional knock-out of two downstream negative regulators of TrkB signaling, Pten (phosphatase with tensin homolog) and Nf1 (neurofibromatosis type 1), enhanced filopodial motility. This effect was PI3K-dependent and correlated with synaptic density. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was preferentially localized in filopodia and this distribution was enhanced by BDNF application. Thus, intracellular control of filopodial dynamics converged on PI3K activation and PIP3 accumulation, a cellular paradigm conserved for chemotaxis in other cell types. Our results suggest that filopodial movement is not random, but responsive to synaptic guidance molecules.

In vivo role of truncated trkb receptors during sensory ganglion neurogenesis

The mammalian trkB locus undergoes alternative splicing to produce two different types of brain-derived neurotrophic factor receptors. The first type is the full-length receptor tyrosine kinase (TrkB(Tk+); the second type is a truncated receptor lacking the intracellular tyrosine kinase domain (TrkB(Tk-)). To investigate the function of both types of TrkB receptor in vivo, we have generated knockout mice lacking all isoforms of the TrkB receptor (trkB-/-) and compared sensory neuron survival in these mice to that in the previously described TrkB kinase domain knockout mice (trkB(k)-/-). We observed that the presence of truncated TrkB receptors in trkB(k)-/- mice results in more severe sensory neuron losses. Increased neuron losses associated with the presence of truncated TrkB were most severe in regions where neuron survival is most dependent on brain-derived neurotrophic factor and neurotrophin-3. Our data suggest that truncated TrkB receptors negatively influence neuron survival by interfering with the function of catalytic TrkB receptors.