Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) that supply presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is largely unknown. I will discuss our recent advances in our understanding of how key components of the presynaptic machinery for neurotransmitter release are transported and assembled focussing on our studies in genome-engineered human induced pluripotent stem cell-derived neurons. Specifically, I will focus on the composition and cell biological identity of the axonal transport vesicles that shuttle key components of neurotransmission to nascent synapses and on machinery for axonal transport and its control by signaling lipids. Our studies identify a crucial mechanism mediating the delivery of SV and active zone proteins to developing synapses and reveal connections to neurological disorders. In the second part of my talk, I will discuss how exocytosis and endocytosis are coupled to maintain presynaptic membrane homeostasis. I will present unpublished data regarding the role of membrane tension in the coupling of exocytosis and endocytosis at synapses. We have identified an endocytic BAR domain protein that is capable of sensing alterations in membrane tension caused by the exocytotic fusion of SVs to initiate compensatory endocytosis to restore plasma membrane area. Interference with this mechanism results in defects in the coupling of presynaptic exocytosis and SV recycling at human synapses.

Alpha synuclein and Lrrk2 are key players in Parkinson's disease and related disorders, but their normal role has been confusing and controversial. Data from acute gene-editing based knockdown, followed by functional assays, will be presented.

Behavior and cognition depend on the integrated action of neural structures and populations distributed throughout the brain. We recently developed a set of molecular imaging tools that enable multiregional processing and plasticity in neural networks to be studied at a brain-wide scale in rodents and nonhuman primates. Here we will describe how a novel genetically encoded activity reporter enables information flow in virally labeled neural circuitry to be monitored by fMRI. Using the reporter to perform functional imaging of synaptically defined neural populations in the rat somatosensory system, we show how activity is transformed within brain regions to yield characteristics specific to distinct output projections. We also show how this approach enables regional activity to be modeled in terms of inputs, in a paradigm that we are extending to address circuit-level origins of functional specialization in marmoset brains. In the second part of the talk, we will discuss how another genetic tool for MRI enables systematic studies of the relationship between anatomical and functional connectivity in the mouse brain. We show that variations in physical and functional connectivity can be dissociated both across individual subjects and over experience. We also use the tool to examine brain-wide relationships between plasticity and activity during an opioid treatment. This work demonstrates the possibility of studying diverse brain-wide processing phenomena using molecular neuroimaging.

The development of the iPS cell technology has revolutionized our ability to study development and diseases in defined in vitro cell culture systems. The talk will focus on Rett Syndrome and discuss two topics: (i) the use of gene editing as an approach to therapy and (ii) the role of MECP2 in gene expression (i) The mutation of the X-linked MECP2 gene is causative for the disease. In a female patient, every cell has a wt copy that is, however, in 50% of the cells located on the inactive X chromosome. We have used epigenetic gene editing tools to activate the wt MECP2 allele on the inactive X chromosome. (ii) MECP2 is thought to act as repressor of gene expression. I will present data which show that MECP2 binds to Pol II and acts as an activator for thousands of genes. The target genes are significantly enriched for Autism related genes. Our data challenge the established model of MECP2’s role in gene expression and suggest novel therapeutic approaches.

Neurodevelopmental disorders (NDDs) impose a global burden, affecting an increasing number of individuals. While some causative genes have been identified, understanding the human-specific mechanisms involved in these disorders remains limited. Traditional gene-driven approaches for modeling brain diseases have failed to capture the diverse and convergent mechanisms at play. Centrosomes and cilia act as intermediaries between environmental and intrinsic signals, regulating cellular behavior. Mutations or dosage variations disrupting their function have been linked to brain formation deficits, highlighting their importance, yet their precise contributions remain largely unknown. Hence, we aim to investigate whether the centrosome/cilia axis is crucial for brain development and serves as a hub for human-specific mechanisms disrupted in NDDs. Towards this direction, we first demonstrated species-specific and cell-type-specific differences in the cilia-genes expression during mouse and human corticogenesis. Then, to dissect their role, we provoked their ectopic overexpression or silencing in the developing mouse cortex or in human brain organoids. Our findings suggest that cilia genes manipulation alters both the numbers and the position of NPCs and neurons in the developing cortex. Interestingly, primary cilium morphology is disrupted, as we find changes in their length, orientation and number that lead to disruption of the apical belt and altered delamination profiles during development. Our results give insight into the role of primary cilia in human cortical development and address fundamental questions regarding the diversity and convergence of gene function in development and disease manifestation. It has the potential to uncover novel pharmacological targets, facilitate personalized medicine, and improve the lives of individuals affected by NDDs through targeted cilia-based therapies.

Astrocytes are multifunctional glial cells, implicated in neurogenesis and synaptogenesis, supporting and fine-tuning neuronal activity and maintaining brain homeostasis by controlling blood-brain barrier permeability. During the last years a number of studies have shown that astrocytes can also be converted into neurons if they force-express neurogenic transcription factors or miRNAs. Direct astrocytic reprogramming to induced-neurons (iNs) is a powerful approach for manipulating cell fate, as it takes advantage of the intrinsic neural stem cell (NSC) potential of brain resident reactive astrocytes. To this end, astrocytic cell fate conversion to iNs has been well-established in vitro and in vivo using combinations of transcription factors (TFs) or chemical cocktails. Challenging the expression of lineage-specific TFs is accompanied by changes in the expression of miRNAs, that post-transcriptionally modulate high numbers of neurogenesis-promoting factors and have therefore been introduced, supplementary or alternatively to TFs, to instruct direct neuronal reprogramming. The neurogenic miRNA miR-124 has been employed in direct reprogramming protocols supplementary to neurogenic TFs and other miRNAs to enhance direct neurogenic conversion by suppressing multiple non-neuronal targets. In our group we aimed to investigate whether miR-124 is sufficient to drive direct reprogramming of astrocytes to induced-neurons (iNs) on its own both in vitro and in vivo and elucidate its independent mechanism of reprogramming action. Our in vitro data indicate that miR-124 is a potent driver of the reprogramming switch of astrocytes towards an immature neuronal fate. Elucidation of the molecular pathways being triggered by miR-124 by RNA-seq analysis revealed that miR-124 is sufficient to instruct reprogramming of cortical astrocytes to immature induced-neurons (iNs) in vitro by down-regulating genes with important regulatory roles in astrocytic function. Among these, the RNA binding protein Zfp36l1, implicated in ARE-mediated mRNA decay, was found to be a direct target of miR-124, that be its turn targets neuronal-specific proteins participating in cortical development, which get de-repressed in miR-124-iNs. Furthermore, miR-124 is potent to guide direct neuronal reprogramming of reactive astrocytes to iNs of cortical identity following cortical trauma, a novel finding confirming its robust reprogramming action within the cortical microenvironment under neuroinflammatory conditions. In parallel to their reprogramming properties, astrocytes also participate in the maintenance of blood-brain barrier integrity, which ensures the physiological functioning of the central nervous system and gets affected contributing to the pathology of several neurodegenerative diseases. To study in real time the dynamic physical interactions of astrocytes with brain vasculature under homeostatic and pathological conditions, we performed 2-photon brain intravital imaging in a mouse model of systemic neuroinflammation, known to trigger astrogliosis and microgliosis and to evoke changes in astrocytic contact with brain vasculature. Our in vivo findings indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, however this event is at compensated by the cross-talk of astrocytes with activated microglia, safeguarding blood vessel coverage and maintenance of blood-brain integrity.

Microglia are the resident CNS macrophages of the brain parenchyma. They have many and opposing roles in health and disease, ranging from inflammatory to anti-inflammatory and protective functions, depending on the developmental stage and the disease context. In Multiple Sclerosis, microglia are involved to important hallmarks of the disease, such as inflammation, demyelination, axonal damage and remyelination, however the exact mechanisms controlling their transformation towards a protective or devastating phenotype during the disease progression remains largely unknown until now. We wish to understand how brain microglia respond to demyelinating insults and how their behaviour changes in recovery. To do so we developed a novel histopathological analysis approach in 3D and a cell-based analysis tool that when applied in the cuprizone model of demyelination revealed region- and disease- dependent changes in microglial dynamics in the brain grey matter during demyelination and remyelination. We now use similar approaches with the aim to unravel sensitive changes in microglial dynamics during neuroinflammation in the EAE model. Furthermore, we employ constitutive knockout and tamoxifen-inducible gene-targeting approaches, immunological techniques, genetics and bioinformatics and currently seek to clarify the specific role of the brain resident microglial NF-κB molecular pathway versus other tissue macrophages in EAE.

Sensory neurons of the vagus nerve monitor distention and stretch in the gastrointestinal tract. We used genetically guided anatomical tracing, optogenetics and electrophysiology to identify and characterize two vagal sensory neuronal subtypes expressing Prox2 and Runx3. We show that these neuronal subtypes innervate the esophagus where they display regionalized innervation patterns. Electrophysiological analyses showed that they are both low threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis and swallowing in freely behaving animals. Our work reveals the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders.

Although bradykinesia, tremor, and rigidity are hallmark motor defects in Parkinson’s disease (PD) patients, they also experience motor learning impairments and non-motor symptoms such as depression. The neural basis for these different PD symptoms are not well understood. While current treatments are effective for locomotion deficits in PD, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking. We found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN), and nucleus accumbens (NAc). While PF-->CPu and PF-->STN circuits are critical for locomotion and motor learning respectively, inhibition of the PF-->NAc circuit induced a depression-like state. While chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation at PF-->STN synapses restored motor learning behavior in PD model mice. Furthermore, activation of NAc-projecting PF neurons rescued depression-like PD phenotypes. Importantly, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

SCN8A encodes a major voltage-gated sodium channel expressed in CNS and PNS neurons.  Gain-of-function and loss-of-function mutations contribute to  human disorders, most notably Developmental and Epileptic Encephalophy (DEE). More than 600 affected individuals have been reported, with the most common  mechanism of de novo, gain-of-function mutations.  We have developed constitutive  and conditional models of gain- and loss- of function mutations in the mouse and  characterized the effects of on neuronal firing and neurological phenotypes.  Using CRE lines with cellular and developmental specificity, we have probed the effects of activating  mutant alleles in various classes of neurons in the developing and adult mouse.   Most recently, we are testing genetic therapies that reduce the expression  of gain-of-function mutant alleles.  We are comparing the effectiveness of allele specific  oligos (ASOs), viral delivery of shRNAs, and allele-specific targeting of mutant alleles  using Crispr/Cas9 in mouse models of DEE.

The cerebral cortex contains an extraordinary diversity of excitatory projection neuron (PN) and inhibitory interneurons (IN), wired together to form complex circuits. Spatiotemporally coordinated execution of intrinsic molecular programs by PNs and INs and activity-dependent processes, contribute to cortical development and cortical microcircuits formation. Alterations of these delicate processes have often been associated to neurological/neurodevelopmental disorders. However, despite the groundbreaking discovery that spontaneous activity in the embryonic brain can shape regional identities of distinct cortical territories, it is still unclear whether this early activity contributes to define subtype-specific neuronal fate as well as circuit assembly. In this study, we combined in utero genetic perturbations via CRISPR/Cas9 system and pharmacological inhibition of selected ion channels with RNA-sequencing and live imaging technologies to identify the activity-regulated processes controlling the development of different cortical PN classes, their wiring and the acquisition of subtype specific features. Moreover, we generated human induced pluripotent stem cells (iPSCs) form patients affected by a severe, rare and untreatable form of developmental epileptic encephalopathy. By differentiating cortical organoids form patient-derived iPSCs we create human models of early electrical alterations for studying molecular, structural and functional consequences of the genetic mutations during cortical development. Our ultimate goal is to define the activity-conditioned processes that physiologically occur during the development of cortical circuits, to identify novel therapeutical paths to address the pathological consequences of neonatal epilepsies.

Cells preferentially responding to visual motion in a particular direction are said to be direction-selective, and these were first identified in the primary visual cortex. Since then, direction-selective responses have been observed in the retina of several species, including mice, indicating motion analysis begins at the earliest stage of the visual hierarchy. Yet little is known about how retinal direction selectivity contributes to motion processing in the visual cortex. In this talk, I will present our experimental efforts to narrow this gap in our knowledge. To this end, we used genetic approaches to disrupt direction selectivity in the retina and mapped neuronal responses to visual motion in the visual cortex of mice using intrinsic signal optical imaging and two-photon calcium imaging. In essence, our work demonstrates that direction selectivity computed at the level of the retina causally serves to establish specialized motion responses in distinct areas of the mouse visual cortex. This finding thus compels us to revisit our notions of how the brain builds complex visual representations and underscores the importance of the processing performed in the periphery of sensory systems.

Expression of Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) on excitatory hippocampal neurons in the hippocampus represents a potential new therapeutic strategy for drug-resistant epilepsy. During my talk I will demonstrate that we obtained potent suppression of spontaneous epileptic seizures in mouse and a rat models for temporal lobe epilepsy using different DREADD ligands, up to one year after viral vector expression. The chemogenetic approach clearly outperforms the seizure-suppressing efficacy of currently existing anti-epileptic drugs. Besides the promises, I will also present some of the challenges associated with a potential chemogenetic therapy, including constitutive DREADD activity, tolerance effects, risk for toxicity, paradoxical excitatory effects in non-epileptic hippocampal tissue.

The gradual accumulation of hyperphosphorylated forms of the Tau protein (pTau) in the human brain correlate with cognitive dysfunction and neurodegeneration. I will present our recent findings on the consequences of human pTau aggregation in the hippocampal formation of a mouse tauopathy model. We show that pTau preferentially accumulates in deep-layer pyramidal neurons, leading to their neurodegeneration. In aged but not younger mice, pTau spreads to oligodendrocytes. During ‘goal-directed’ navigation, we detect fewer high-firing pyramidal cells, but coupling to network oscillations is maintained in the remaining cells. The firing patterns of individually recorded and labelled pyramidal and GABAergic neurons are similar in transgenic and non-transgenic mice, as are network oscillations, suggesting intact neuronal coordination. This is consistent with a lack of pTau in subcortical brain areas that provide rhythmic input to the cortex. Spatial memory tests reveal a reduction in short-term familiarity of spatial cues but unimpaired spatial working and reference memory. These results suggest that preserved subcortical network mechanisms compensate for the widespread pTau aggregation in the hippocampal formation. I will also briefly discuss ideas on the subcortical origins of spatial memory and the concept of the cortex as a monitoring device.

Normative development in adolescence indicates that the prefrontal cortex is still under development thereby unable to exert efficient top-down inhibitory control on subcortical regions such as the basolateral amygdala and the nucleus accumbens. This imbalance in the developmental trajectory between cortical and subcortical regions is implicated in expression of the prototypical impulsive, compulsive, reward seeking and risk-taking adolescent behavior. Here we demonstrate that a chronic mild unpredictable stress procedure during adolescence in male Wistar rats arrests the normal behavioral maturation such that they continue to express adolescent-like impulsive, hyperactive, and compulsive behaviors into late adulthood. This arrest in behavioral maturation is associated with the hypoexcitability of prelimbic cortex (PLC) pyramidal neurons and reduced PLC-mediated synaptic glutamatergic control of BLA and nucleus accumbens core (NAcC) neurons that lasts late into adulthood. At the same time stress exposure in adolescence results in the hyperexcitability of the BLA pyramidal neurons sending stronger glutamatergic projections to the NAcC. Chemogenetic reversal of the PLC hypoexcitability decreased compulsivity and improved the expression of goal-directed behavior in rats exposed to stress during adolescence, suggesting a causal role for PLC hypoexcitability in this stress-induced arrested behavioral development. (https://www.biorxiv.org/content/10.1101/2021.11.21.469381v1.abstract)

N-methyl-D-aspartate receptors (NMDARs) are excitatory glutamate-gated ion channels that are expressed throughout the central nervous system. NMDARs mediate calcium entry into cells, and are involved in a host of neurological functions. The GluN2A subunit, encoded by the GRIN2A gene, is expressed by both excitatory and inhibitory neurons, with well described roles in pyramidal cells. By using Grin2a knockout mice, we show that the loss of GluN2A signaling impacts parvalbumin-positive (PV) GABAergic interneuron function in hippocampus. Grin2a knockout mice have 33% more PV cells in CA1 compared to wild type but similar cholecystokinin-positive cell density. Immunohistochemistry and electrophysiological recordings show that excess PV cells do eventually incorporate into the hippocampal network and participate in phasic inhibition. Although the morphology of Grin2a knockout PV cells is unaffected, excitability and action-potential firing properties show age-dependent alterations. Preadolescent (P20-25) PV cells have an increased input resistance, longer membrane time constant, longer action-potential half-width, a lower current threshold for depolarization-induced block of action-potential firing, and a decrease in peak action-potential firing rate. Each of these measures are corrected in adulthood, reaching wild type levels, suggesting a potential delay of electrophysiological maturation. The circuit and behavioral implications of this age-dependent PV interneuron malfunction are unknown. However, neonatal Grin2a knockout mice are more susceptible to lipopolysaccharide and febrile-induced seizures, consistent with a critical role for early GluN2A signaling in development and maintenance of excitatory-inhibitory balance. These results could provide insights into how loss-of-function GRIN2A human variants generate an epileptic phenotypes.

Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and neural circuit remodeling in the hippocampus resulting in increased susceptibility to spontaneous seizures and cognitive dysfunction. Targeting these cascades could prevent or reverse symptom progression and has the potential to provide viable disease-modifying treatments that could reduce the portion of TLE patients (>30%) not responsive to current medical therapies. Changes in GABA(A) receptor subunit expression have been implicated in the pathogenesis of TLE, and the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been shown to be a key regulator of these changes. The JAK/STAT pathway is known to be involved in inflammation and immunity, and to be critical for neuronal functions such as synaptic plasticity and synaptogenesis. Our laboratories have shown that a STAT3 inhibitor, WP1066, could greatly reduce the number of spontaneous recurrent seizures (SRS) in an animal model of pilocarpine-induced status epilepticus (SE). This suggests promise for JAK/STAT inhibitors as disease-modifying therapies, however, the potential adverse effects of systemic or global CNS pathway inhibition limits their use. Development of more targeted therapeutics will require a detailed understanding of JAK/STAT-induced epileptogenic responses in different cell types. To this end, we have developed a new transgenic line where dimer-dependent STAT3 signaling is functionally knocked out (fKO) by tamoxifen-induced Cre expression specifically in forebrain excitatory neurons (eNs) via the Calcium/Calmodulin Dependent Protein Kinase II alpha (CamK2a) promoter. Most recently, we have demonstrated that STAT3 KO in excitatory neurons (eNSTAT3fKO) markedly reduces the progression of epilepsy (SRS frequency) in the intrahippocampal kainate (IHKA) TLE model and protects mice from kainic acid (KA)-induced memory deficits as assessed by Contextual Fear Conditioning. Using data from bulk hippocampal tissue RNA-sequencing, we further discovered a transcriptomic signature for the IHKA model that contains a substantial number of genes, particularly in synaptic plasticity and inflammatory gene networks, that are down-regulated after KA-induced SE in wild-type but not eNSTAT3fKO mice. Finally, we will review data from other models of brain injury that lead to epilepsy, such as TBI, that implicate activation of the JAK/STAT pathway that may contribute to epilepsy development.

Activation of pro-degenerative protein SARM1 in response to diverse physical and disease-relevant injuries triggers programmed axon death. Original studies indicated substantially decreased levels of SARM1 were required for neuroprotection. However, we demonstrate that lowering SARM1 levels by 50% in Sarm1 haploinsufficient mice delays axon degeneration in vivo (after sciatic nerve transection), in vitro (in response to diverse traumatic, neurotoxic, and genetic triggers), and partially prevents neurite outgrowth defects in mice lacking pro-survival factor NMNAT2. We also demonstrate the capacity for Sarm1 antisense oligonucleotides to decrease SARM1 levels by more than 50% which delays or prevents programmed axon degeneration in vitro. Combining Sarm1 haploinsufficiency with antisense oligonucleotides further decreases SARM1 levels and prolongs protection after neurotoxic injuries. These data demonstrate that axon protection occurs in a Sarm1 gene-dose responsive manner and that SARM1 lowering agents have therapeutic potential. Thus, antisense oligonucleotide targeting of Sarm1 is a promising therapeutic strategy against diverse triggers of axon degeneration.

Pathogenic variants in HCN1 are associated with severe developmental and epileptic encephalopathies (DEE). We have engineered the Hcn1 M294L heterozygous knock-in (Hcn1M294L) mouse which is a homolog of the de novo HCN1 M305L recurrent pathogenic variant. The mouse recapitulates the phenotypic features of patients including having spontaneous seizures and a learning deficit. In this talk I will present experimental work that probes the molecular and cellular mechanisms underlying hyper-excitability in the mouse model. This will include testing the efficacy of currently available antiepileptic drugs and a novel precision medicine approach. I will also briefly touch on how disease biology can give insights into the biophysical properties of HCN channels.

Tracking heading within an environment is a fundamental requirement for flexible, goal-directed navigation. In insects, a head-direction representation that guides the animal’s movements is maintained in a conserved brain region called the central complex. Two-photon calcium imaging of genetically targeted neural populations in the central complex of tethered fruit flies behaving in virtual reality (VR) environments has shown that the head-direction representation is updated based on self-motion cues and external sensory information, such as visual features and wind direction. Thus far, the head direction representation has mainly been studied in VR settings that only give flies control of the angular rotation of simple sensory cues. How the fly’s head direction circuitry enables the animal to navigate in dynamic, immersive and naturalistic environments is largely unexplored. I have developed a novel setup that permits imaging in complex VR environments that also accommodate flies’ translational movements. I have previously demonstrated that flies perform visually-guided navigation in such an immersive VR setting, and also that they learn to associate aversive optogenetically-generated heat stimuli with specific visual landmarks. A stable head direction representation is likely necessary to support such behaviors, but the underlying neural mechanisms are unclear. Based on a connectomic analysis of the central complex, I identified likely circuit mechanisms for prioritizing and combining different sensory cues to generate a stable head direction representation in complex, multimodal environments. I am now testing these predictions using calcium imaging in genetically targeted cell types in flies performing 2D navigation in immersive VR.