Altered migration of forebrain inhibitory interneurons in a mouse model of intellectual disability

OPHN1 is a X-linked gene associated to intellectual disability (ID) that encodes a Rho-GTPase activating protein (RhoGAP) that regulates the conformational rearrangements of actin filaments involved in several developmental processes including axon outgrowth, dendritic maturation and cell migration. The impact of OPHN1 mutation on neuronal morphology and function has been studied mostly in glutamatergic neurons. GABAergic inhibitory interneurons exert an important role in in the excitation inhibition balance of neuronal network. Indeed alterations in development and function of GABAergic interneurons have been associated to several neurodevelopmental disorders. Whether and how OPHN1 affects inhibitory circuitry remains largely to be understood. To address this question, I have been studying adult neurogenesis of inhibitory interneurons in the subventricular zone (SVZ) in a mouse model of intellectual disability, i.e. a line of mice carrying a null mutation for OPHN1. In the subventricular zone (SVZ), along the wall of the lateral ventricles, neurogenesis of interneurons continues throughout life. From the SVZ neuronal precursors migrate along the rostral migratory stream to reach the olfactory bulb (OB) where they become fully mature interneurons. Noteworthy, in mammals, including human infants, the SVZ-derived inhibitory interneurons migrate not only to the OB but also to several subcortical and cortical areas. These postnatally generated interneurons are thought to play a prominent role in the postnatal development and plasticity of the brain. In a previous work we found that loss-of-function mutation of OPHN1 did not affect the generation of neuronal precursors in the subventricular zone. However, the number of adult-born cells that reached the olfactory bulb was dramatically reduced in OPHN1-/y mice. These results suggested that OPHN1 mutation could affect neuronal migration. We explored whether and how the migration was altered in OPHN1-/y mice. Combining birthdating experiments (i.e. using BrdU as a marker of new cells) and quantitative anatomy we studied the distribution, the morphology and the directionality of the adult born cells along the rostral migratory stream (RMS). Remarkably, we found that the distribution of neuronal precursors along the rostral migratory stream was deeply altered in OPHN1-/y mice, with most of the newly generated cells clamped in the initial portion of the RMS, and only very few cells in the final portion of the RMS and the OB in OPHN1-/y mice respect to controls. To dissect the mechanism underlying altered cell migration, we performed time-lapse imaging of migrating neuroblasts on thick sagittal sections of the brain by means of two-photon imaging. We investigated the velocity and the directionality of migrating cells, in wt and OPHN1-/y mice. We found that the speed of neuroblasts progression was significantly reduced in OPHN1-/y mice respect to controls. Furthermore the percentage of cells migrating toward the OB (directionality) was significantly reduced in OPHN1-/y with respect to controls. GABA is known to modulate neuroblast migration during development. GABA, abundantly present in the RMS, is thought to be released from neuroblasts and act in autocrine and paracrine fashion on neuroblasts that express GABAA receptors. Accumulation of ambient GABA is prevented by GABA uptake by astrocyte-like cells, which ensheath the chains of migrating precursors. GABA is the major inhibitory neurotransmitter in adult brain. GABA response polarity (hyperpolarization versus depolarization) is however, not univocal, but it is critically regulated by intracellular chloride concentration, that is in turn modulated by specific co-transtranporters, NKCC1 and KCC2. NKCC1, mostly expressed in immature neurons, favors high intracellular chloride concentration leading to depolarizing GABA. KCC2, widely expressed in mature neurons, favors low intracellular chloride concentration, leading to hyperpolarizing GABA. Depolarizing GABA is thought to play a prominent role in several developmental processes, including cell migration. To assess how GABA modulates cell migration in OPHN1-/y mice, time-lapse imaging of neuronal precursors was performed in baseline conditions and in presence of GABA in control and in OPHN1-/y mice. GABA application reduced speed of migration in wt mice, but exerted an opposite effect in OPHN1-/y mice, resulting in an increased speed of migration in OPHN1-/y mice. The inhibition of GABAA receptors upon bicuculline application increased the speed of neuroblasts progression in wt mice, while in OPHN1-/y mice bicuculline application reduced the overall speed of migrating SVZ-derived cells. We explored the effect of NKCC1 blocker, bumetanide, on the motility of the migrating cells. As immature neurons, migrating cells expressed mostly NKCC1. In presence of NKCC1 blocker bumetanide, the speed of newly generated cells was reduced in wt mice. However, no effect was observed on the motility of migrating cells in OPHN1-/y mice. We investigated the effects of KCC2 inhibition in migrating precursors. In wt mice, the application of the KCC2 inhibitor VU0463271 did not affect neuroblast motility. By contrast, blocking KCC2 resulted in a significant increase in the translocation speed in OPHN1-/y mice. These findings corroborated the hypothesis that the polarity of GABA response is altered in migrating neuroblasts along the RMS in OPHN1-/y mice. Noteworthy, none of the drug used affected the directionality of the migrating cells in controls, nor in OPHN1-/y mice. In a previous work in our lab, we found that the chronic administration of the ROCK non-competitive inhibitor fasudil rescued the number of adult-born interneurons in the OB of OPHN1-/y mice with respect to littermate controls. We therefore tested the effect of acute application of fasudil on slices. Interestingly, we found that fasudil completely rescued the directionality but it did not affect the progression of migrating cells in OPHN1-/y versus controls. As mentioned above, in a previous work in our lab we found that chronic treatment with fasudil administered to mice ad libitum in the drinking water rescued the number of newborn GABAergic interneurons in the OB 15 days after their birth in the SVZ, when all the cells reached their target layer in the OB. Given the effects of acute fasudil on the directionality, but not on the speed, of migrating cells in OPHN1-/y mice, we asked whether chronic treatment with fasudil could result in a delayed arrival of the whole complement of newly generated cells in the OB. To verify this hypothesis we quantified the complement of newly generated cells that reach the OB 9 days after their generation in controls and in OPHN1-/y treated mice. In controls the number of newly generated cells that reach the OB at 9 and 15 dpi is similar. We found that at 9 days after their generation in the SVZ, the number of newly generated cells that reached the OB was still significantly lower in OPHN1-/y mice treated with fasudil with respect to controls. In agreement with our data in slices, the effect of fasudil, in vivo, rescued the directionality, but not the speed of neuronal precursors progression, resulting in a delayed arrival respect to controls. Combining quantitative anatomy and time-lapse two-photon imaging in vivo, we found that loss-of-function mutation of OPHN1 leads to the inability of neuronal precursor cells to migrate properly from the SVZ to the OB. The hampered migration could be ascribed to a subverted GABA signalling or GABA response polarity in migrating cells in OPHN1-/y mice. Null mutation of OPHN1, could hamper neuroblasts motility acting on two different mechanisms governed by two distinct signaling pathways. In addition, this likely alters excitatory/inhibitory balance in the circuitry of the OB, and it may contribute to the pathophysiology of ID.