Verna Louhivuori

Aberrant differentiation of glutamatergic cells in neocortex of mouse model for fragile X syndrome

The lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. Our previous studies revealed alterations in the differentiation of FMRP-deficient neural progenitors. Here, we show abnormalities in neurogenesis in the mouse and human embryonic FMRP-deficient brain as well as after in utero transfection of I304N mutated FMRP, which acts in a dominant negative manner in the wild-type mouse brain. Progenitors accumulated abnormally in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) mouse neocortex. An increased density of cells expressing sequentially an intermediate progenitor marker, T-box transcription factor (Tbr2), and a postmitotic neuron marker, T-brain 1 (Tbr1), indicated that the differentiation to glutamatergic cell lineages was particularly disturbed. These abnormalities were associated with an increased density of pyramidal cells of the layer V in the early postnatal neocortex suggesting a role for FMRP in the regulation of the differentiation of neocortical glutamatergic neurons.

BDNF and TrkB in neuronal differentiation of Fmr1-knockout mouse.

Fragile X syndrome (FXS) is a common cause of inherited mental retardation and the best characterized form of autistic spectrum disorders. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP), which leads to abnormalities in the differentiation of neural progenitor cells (NPCs) and in the development of dendritic spines and neuronal circuits. Brain-derived neurotrophic factor (BDNF) and its TrkB receptors play a central role in neuronal maturation and plasticity. We studied BDNF/TrkB actions in the absence of FMRP and show that an increase in catalytic TrkB expression in undifferentiated NPCs of Fmr1-knockout (KO) mice, a mouse model for FXS, is associated with changes in the differentiation and migration of neurons expressing TrkB in neurosphere cultures and in the developing cortex. Aberrant intracellular calcium responses to BDNF and ATP in subpopulations of differentiating NPCs combined with changes in the expression of BDNF and TrkB suggest cell subtype-specific alterations during early neuronal maturation in the absence of FMRP. Furthermore, we show that dendritic targeting of Bdnf mRNA was increased under basal conditions and further enhanced in cortical layer V and hippocampal CA1 neurons of Fmr1-KO mice by pilocarpine-induced neuronal activity represented by convulsive seizures, suggesting that BDNF/TrkB-mediated feedback mechanisms for strengthening the synapses were compromised in the absence of FMRP. Pilocarpine-induced seizures caused an accumulation of Bdnf mRNA transcripts in the most proximal segments of dendrites in cortical but not in hippocampal neurons of Fmr1-KO mice. In addition, BDNF protein levels were increased in the hippocampus but reduced in the cortex of Fmr1-KO mice in line with regional differences of synaptic plasticity in the brain of Fmr1-KO mice. Altogether, the present data suggest that alterations in the BDNF/TrkB signaling modulate brain development and impair synaptic plasticity in FXS.

Reduction of BDNF expression in Fmr1 knockout mice worsens cognitive deficits but improves hyperactivity and sensorimotor deficits

Fragile X syndrome (FXS) is a common cause of inherited intellectual disability and a well‐characterized form of autism spectrum disorder. As brain‐derived neurotrophic factor (BDNF) is implicated in the pathophysiology of FXS we examined the effects of reduced BDNF expression on the behavioral phenotype of an animal model of FXS, Fmr1 knockout (KO) mice, crossed with mice carrying a deletion of one copy of the Bdnf gene (Bdnf+/−). Fmr1 KO mice showed age‐dependent alterations in hippocampal BDNF expression that declined after the age of 4 months compared to wild‐type controls. Mild deficits in water maze learning in Bdnf+/− and Fmr1 KO mice were exaggerated and contextual fear learning significantly impaired in double transgenics. Reduced BDNF expression did not alter basal nociceptive responses or central hypersensitivity in Fmr1 KO mice. Paradoxically, the locomotor hyperactivity and deficits in sensorimotor learning and startle responses characteristic of Fmr1 KO mice were ameliorated by reducing BNDF, suggesting changes in simultaneously and in parallel working hippocampus‐dependent and striatum‐dependent systems. Furthermore, the obesity normally seen in Bdnf+/− mice was eliminated by the absence of fragile X mental retardation protein 1 (FMRP). Reduced BDNF decreased the survival of newborn cells in the ventral part of the hippocampus both in the presence and absence of FMRP. Since a short neurite phenotype characteristic of newborn cells lacking FMRP was not found in cells derived from double mutant mice, changes in neuronal maturation likely contributed to the behavioral phenotype. Our results show that the absence of FMRP modifies the diverse effects of BDNF on the FXS phenotype.

Neurotransmitter responsiveness during early maturation of neural progenitor cells.

Neurotransmitters are potential regulators of proliferation and differentiation of neural progenitor cells (NPC). To gain insight into the dynamics of neurotransmitter responsiveness, neurospheres were prepared from the lateral ventricles of postnatal day 6/7 mice. Individual NPCs migrating out from spheres were simultaneously monitored using Ca(2+) imaging, during the initial 8 days of differentiation, at an area between the inner edge of the sphere and outer periphery of the area of migration. At the first day of differentiation most cells showed metabotropic responses (Ca(2+) discharge from stores) to glutamate (pharmacologically identified as metabotropic glutamate receptor 5, mGluR 5), norepinephrine (NE), acetylcholine (Ach) and ATP, and a smaller proportion of cells also responded to substance P (SP). When outside the neurosphere, many of mGluR5 responding cells gained immunostaining for markers of neuronal lineage (Tuj-1 and NeuN). The number of cells responding through mGluR5 (and responses to Ach, NE and SP) showed during subsequent days of differentiation (day 2-3 onwards) a decline with time and progressively disappeared at the outer periphery of the area of migration. Conversely the number ionotropic glutamate responses as well as responses to depolarization increased in this area. After 5-8 days of differentiation mGluR5 responses could only be observed at the very inner edge of the neurosphere. At 8 days the migrated cells showed very robust ionotropic responses to glutamate, NMDA and depolarization comparable to mature neurons. Taken together, the data presented here suggest that differentiation of NPCs is a dynamic process triggered by cell migration, which leads to a loss of regulatory influences imposed by the inner milieu of the neurosphere. The subsequent switch or loss of metabotropic responses to glutamate, SP, NE, Ach and ATP with the gain of excitable characteristics such as ionotropic responses appears to be a key event in the final differentiation process.

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non‐selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR‐32 undergoes a remarkable differentiation in response to treatment with 5‐bromo‐2‐deoxyuridine. The cells acquire a neuronal morphology with increased expression of N‐type voltage gated calcium channels and neurotransmitters. Here we show using RT‐PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR‐32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl‐isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC‐030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR‐32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR‐32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67–74, 2009.

The Val66Met polymorphism in the BDNF gene is associated with epilepsy in fragile X syndrome

The Val66Met polymorphism in the brain-derived neurotrophic factor (BDNF) gene may modulate the epilepsy phenotype. We investigated the impact of polymorphisms in the BDNF gene on clinical features in fragile X syndrome (FXS). In our study sample, the Met66 allele associated with epilepsy of finnish FXS men. Abnormalities in BDNF-mediated plasticity are shown in FXS and the present data suggest that the Met66 allele might predispose FXS males to epilepsy.

Brain-derived neurotrophic factor increases the motility of a particular N-methyl-d-aspartate /GABA-responsive subset of neural progenitor cells

Neurotrophins like brain-derived neurotrophic factor (BDNF) promote the migration of subsets of neural progenitor cells. The mechanism by which motility is increased and the functional properties of BDNF-responsive cells are not very well known. We have used the neurosphere model, combining time-lapse microscopy, immunocytochemistry, and Ca(2+) imaging, to study the effect of BDNF on parameters such as motility and neurotransmitter responsiveness of migrating neural progenitors. At the initiation of differentiation thick glial glutamate-aspartate transporter (GLAST)-positive radial processes emerged from the neurosphere, followed by the exit of neuron-like cells. The neuron-like cells moved outside the radial processes in a phasic manner with intermittent surges of motility and stationary periods. BDNF increased the number and promoted the progress of the neuron-like cells by prolonging surges and decreasing the length of stationary phases. The average rate of cellular movement during surges was unaffected by BDNF. BDNF also caused a several fold increase in positive staining for tropomyosin-related kinase B (TrkB) receptors and neuronal markers such as Calbindin, microtubule-associated protein-2 (MAP-2), and neuron-specific nuclear protein (NeuN) in cells outside the radial network. Calcium imaging allowed for further characterization of the BDNF-responsive cell population. Kainate-responsive cells, denoting the expression of AMPA/kainate receptors, dominated in the outer migration layers while cells responding to (S)-3,5-dihydroxyphenylglycine (DHPG) via metabotropic glutamate receptor 5 (mGluR5) dominated in the inner migration layers. BDNF did not appreciably affect the distribution of these cells but promoted the redistribution of a small subpopulation (about 20%) of N-methyl-D-aspartate (NMDA)- and GABA-responsive cells to the outermost layers of migration. The results demonstrate that BDNF does not affect cell motility per se but alters the phasic behavior of cell movement by promoting periods of high motility in a defined subpopulation of cells which give a robust Ca(2+) response to NMDA and GABA.

Role of Low Voltage Activated Calcium Channels in Neuritogenesis and Active Migration of Embryonic Neural Progenitor Cells

The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

Effect of glutamate receptor antagonists on migrating neural progenitor cells.

Neurotransmitters such as glutamate are potential regulators of neurogenesis. Interference with defined glutamate receptor subtypes affects proliferation, migration and differentiation of neural progenitor cells. The cellular targets for the actions of different glutamate receptor ligands are less well known. In this study we have combined calcium imaging, measurement of membrane potential, time‐lapse imaging and immunocytochemistry to obtain a spatial overview of migrating mouse embryonic neural progenitor cell‐derived cells responding to glutamate receptor agonists and antagonists. Responses via metabotropic glutamate receptor 5 correlated with radial glial cells and dominated in the inner migration zones close to the neurosphere. Block of metabotropic glutamate receptor 5 resulted in shorter radial glial processes, a transient increase in neuron‐like cells emerging from the neurosphere and increased motility of neuron‐like cells. α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)/kainate receptors are present on the majority of migrating neuronal cells, which with time accumulate at the outer edge of the migration zone. Blocking these receptors leads to an enhanced extension of radial glial processes and a reduced motility of neuron‐like cells. Our results indicate that functional glutamate receptors have profound effects on the motility of neural progenitor cells. The main target for metabotropic glutamate receptor 5 appears to be radial glial cells while AMPA/kainate receptors are mainly expressed in newborn neuronal cells and regulate the migratory progress of these cells. The results suggest that both metabotropic glutamate receptor 5 and AMPA/kainate receptors are of importance for the guidance of migrating embryonic progenitor cells.

Tissue Plasminogen Activator Contributes to Alterations of Neuronal Migration and Activity-Dependent Responses in Fragile X Mice

Fragile X syndrome (FXS) is the most common inherited neurodevelopmental disorder with intellectual disability. Here, we show that the expression of tissue plasminogen activator (tPA) is increased in glial cells differentiated from neural progenitors of Fmr1 knock-out mice, a mouse model for FXS, and that tPA is involved in the altered migration and differentiation of these progenitors lacking FMR1 protein (FMRP). When tPA function is blocked with an antibody, enhanced migration of doublecortin-immunoreactive neurons in 1 d differentiated FMRP-deficient neurospheres is normalized. In time-lapse imaging, blocking the tPA function promotes early glial differentiation and reduces the velocity of nuclear movement of FMRP-deficient radial glia. In addition, we show that enhanced intracellular Ca2+ responses to depolarization with potassium are prevented by the treatment with the tPA-neutralizing antibody in FMRP-deficient cells during early neural progenitor differentiation. Alterations of the tPA expression in the embryonic, postnatal, and adult brain of Fmr1 knock-out mice suggest an important role for tPA in the abnormal neuronal differentiation and plasticity in FXS. Altogether, the results indicate that tPA may prove to be an interesting potential target for pharmacological intervention in FXS.