David Gall

Pyramidal Neurons Derived from Human Pluripotent Stem Cells Integrate Efficiently into Mouse Brain Circuits In Vivo

The study of human cortical development has major implications for brain evolution and diseases but has remained elusive due to paucity of experimental models. Here we found that human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), cultured without added morphogens, recapitulate corticogenesis leading to the sequential generation of functional pyramidal neurons of all six layer identities. After transplantation into mouse neonatal brain, human ESC-derived cortical neurons integrated robustly and established specific axonal projections and dendritic patterns corresponding to native cortical neurons. The differentiation and connectivity of the transplanted human cortical neurons complexified progressively over several months in vivo, culminating in the establishment of functional synapses with the host circuitry. Our data demonstrate that human cortical neurons generated in vitro from ESC/iPSC can develop complex hodological properties characteristic of the cerebral cortex in vivo, thereby offering unprecedented opportunities for the modeling of human cortex diseases and brain repair.

Dopamine D2 and Adenosine A2A Receptors Regulate NMDA-Mediated Excitation in Accumbens Neurons Through A2A-D2 Receptor Heteromerization

Bursting activity of striatal medium spiny neurons results from membrane potential oscillations between a down- and an upstate that could be regulated by G-protein-coupled receptors. Among these, dopamine D2 and adenosine A2A receptors are highly enriched in striatal neurons and exhibit strong interactions whose physiological significance and molecular mechanisms remain partially unclear. More particularly, respective involvements of common intracellular signaling cascades and A2A–D2 receptor heteromerization remain unknown. Here we show, by performing perforated-patch-clamp recordings on brain slices and loading competitive peptides, that D2 and A2A receptors regulate the induction by N-methyl-D-aspartate of a depolarized membrane potential plateau through mechanisms relying upon specific protein–protein interactions. Indeed, D2 receptor activation abolished transitions between a hyperpolarized resting potential and a depolarized plateau potential by regulating the CaV1.3a calcium channel activity through interactions with scaffold proteins Shank1/3. Noticeably, A2A receptor activation had no effect per se but fully reversed the effects of D2 receptor activation through a mechanism in which A2A–D2 receptors heteromerization is strictly mandatory, demonstrating therefore a first direct physiological relevance of these heteromers. Our results show that membrane potential transitions and firing patterns in striatal neurons are tightly controlled by D2 and A2A receptors through specific protein–protein interactions including A2A–D2 receptors heteromerization.

Neurons and cardiomyocytes derived from induced pluripotent stem cells as a model for mitochondrial defects in Friedreich's ataxia

SUMMARY Friedreich’s ataxia (FRDA) is a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy. FRDA is due to expanded GAA repeats within the first intron of the gene encoding frataxin, a conserved mitochondrial protein involved in iron-sulphur cluster biosynthesis. This mutation leads to partial gene silencing and substantial reduction of the frataxin level. To overcome limitations of current cellular models of FRDA, we derived induced pluripotent stem cells (iPSCs) from two FRDA patients and successfully differentiated them into neurons and cardiomyocytes, two affected cell types in FRDA. All FRDA iPSC lines displayed expanded GAA alleles prone to high instability and decreased levels of frataxin, but no biochemical phenotype was observed. Interestingly, both FRDA iPSC-derived neurons and cardiomyocytes exhibited signs of impaired mitochondrial function, with decreased mitochondrial membrane potential and progressive mitochondrial degeneration, respectively. Our data show for the first time that FRDA iPSCs and their neuronal and cardiac derivatives represent promising models for the study of mitochondrial damage and GAA expansion instability in FRDA.

Intracellular calcium regulation by burst discharge determines bidirectional long-term synaptic plasticity at the cerebellum input stage.

Variations in intracellular calcium concentration ([Ca2+]i) provide a critical signal for synaptic plasticity. In accordance with Hebbs postulate (Hebb, 1949), an increase in postsynaptic [Ca2+]i can induce bidirectional changes in synaptic strength depending on activation of specific biochemical pathways (Bienenstock et al., 1982; Lisman, 1989; Stanton and Sejnowski, 1989). Despite its strategic location for signal processing, spatiotemporal dynamics of [Ca2+]i changes and their relationship with synaptic plasticity at the cerebellar mossy fiber (mf)-granule cell (GrC) relay were unknown. In this paper, we report the plasticity/[Ca2+]i relationship for GrCs, which are typically activated by mf bursts (Chadderton et al., 2004). Mf bursts caused a remarkable [Ca2+]i increase in GrC dendritic terminals through the activation of NMDA receptors, metabotropic glutamate receptors (probably acting through IP3-sensitive stores), voltage-dependent calcium channels, and Ca2+-induced Ca2+ release. Although [Ca2+]i increased with the duration of mf bursts, long-term depression was found with a small [Ca2+]i increase (bursts <250 ms), and long-term potentiation (LTP) was found with a large [Ca2+]i increase (bursts >250 ms). LTP and [Ca2+]i saturated for bursts >500 ms and with theta-burst stimulation. Thus, bursting enabled a Ca2+-dependent bidirectional Bienenstock-Cooper-Munro-like learning mechanism providing the cellular basis for effective learning of burst patterns at the input stage of the cerebellum.

Purkinje cell dysfunction and alteration of long-term synaptic plasticity in fetal alcohol syndrome.

In cerebellum and other brain regions, neuronal cell death because of ethanol consumption by the mother is thought to be the leading cause of neurological deficits in the offspring. However, little is known about how surviving cells function. We studied cerebellar Purkinje cells in vivo and in vitro to determine whether function of these cells was altered after prenatal ethanol exposure. We observed that Purkinje cells that were prenatally exposed to ethanol presented decreased voltage-gated calcium currents because of a decreased expression of the γ-isoform of protein kinase C. Long-term depression at the parallel fiber–Purkinje cell synapse in the cerebellum was converted into long-term potentiation. This likely explains the dramatic increase in Purkinje cell firing and the rapid oscillations of local field potential observed in alert fetal alcohol syndrome mice. Our data strongly suggest that reversal of long-term synaptic plasticity and increased firing rates of Purkinje cells in vivo are major contributors to the ataxia and motor learning deficits observed in fetal alcohol syndrome. Our results show that calcium-related neuronal dysfunction is central to the pathogenesis of the neurological manifestations of fetal alcohol syndrome and suggest new methods for treatment of this disorder.

Inactivation of Calcium-Binding Protein Genes Induces 160 Hz Oscillations in the Cerebellar Cortex of Alert Mice

Oscillations in neuronal populations may either be imposed by intrinsically oscillating pacemakers neurons or emerge from specific attributes of a distributed network of connected neurons. Calretinin and calbindin are two calcium-binding proteins involved in the shaping of intraneuronal Ca2+ fluxes. However, although their physiological function has been studied extensively at the level of a single neuron, little is known about their role at the network level. Here we found that null mutations of genes encoding calretinin or calbindin induce 160 Hz local field potential oscillations in the cerebellar cortex of alert mice. These oscillations reached maximum amplitude just beneath the Purkinje cell bodies and are reinforced in the cerebellum of mice deficient in both calretinin and calbindin. Purkinje cells fired simple spikes phase locked to the oscillations and synchronized along the parallel fiber axis. The oscillations reversibly disappeared when gap junctions or either GABAA or NMDA receptors were blocked. Cutaneous stimulation of the whisker region transiently suppressed the oscillations. However, the intrinsic somatic excitability of Purkinje cells recorded in slice preparation was not significantly altered in mutant mice. Functionally, these results suggest that 160 Hz oscillation emerges from a network mechanism combining synchronization of Purkinje cell assemblies through parallel fiber excitation and the network of coupled interneurons of the molecular layer. These findings demonstrate that subtle genetically induced modifications of Ca2+ homeostasis in specific neuron types can alter the observed dynamics of the global network.

Aminopyridines correct early dysfunction and delay neurodegeneration in a mouse model of spinocerebellar ataxia type 1.

The contribution of neuronal dysfunction to neurodegeneration is studied in a mouse model of spinocerebellar ataxia type 1 (SCA1) displaying impaired motor performance ahead of loss or atrophy of cerebellar Purkinje cells. Presymptomatic SCA1 mice show a reduction in the firing rate of Purkinje cells (both in vivo and in slices) associated with a reduction in the efficiency of the main glutamatergic synapse onto Purkinje cells and with increased A-type potassium current. The A-type potassium channel Kv4.3 appears to be internalized in response to glutamatergic stimulation in Purkinje cells and accumulates in presymptomatic SCA1 mice. SCA1 mice are treated with aminopyridines, acting as potassium channel blockers to test whether the treatment could improve neuronal dysfunction, motor behavior, and neurodegeneration. In acutely treated young SCA1 mice, aminopyridines normalize the firing rate of Purkinje cells and the motor behavior of the animals. In chronically treated old SCA1 mice, 3,4-diaminopyridine improves the firing rate of Purkinje cells, the motor behavior of the animals, and partially protects against cell atrophy. Chronic treatment with 3,4-diaminopyridine is associated with increased cerebellar levels of BDNF, suggesting that partial protection against atrophy of Purkinje cells is possibly provided by an increased production of growth factors secondary to the reincrease in electrical activity. Our data suggest that aminopyridines might have symptomatic and/or neuroprotective beneficial effects in SCA1, that reduction in the firing rate of Purkinje cells can cause cerebellar ataxia, and that treatment of early neuronal dysfunction is relevant in neurodegenerative disorders such as SCA1.

Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities

Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV’s putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV−/−) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV+/−) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV−/− and PV+/− mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a common link between apparently unrelated ASD-associated synapse structure/function phenotypes.

BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets

During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signaling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition. We identified Bcl6, an oncogene, as encoding a proneurogenic factor that is required for proper neurogenesis of the mouse cerebral cortex. BCL6 promoted the neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. BCL6 triggered exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD+-dependent deacetylase Sirt1, which was required for BCL6-dependent neurogenesis. The resulting epigenetic silencing of Hes5 led to neuronal differentiation despite active Notch signaling. Our findings suggest a role for BCL6 in neurogenesis and uncover Notch-BCL6-Sirt1 interactions that may affect other aspects of physiology and disease.

Matrix-binding vascular endothelial growth factor (VEGF) isoforms guide granule cell migration in the cerebellum via VEGF receptor Flk1

Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.