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The optogenetic approach to gain control over neuronal excitability both in vitro and in vivo has emerged as a fascinating scientific tool to explore neuronal networks, but it also opens possibilities for developing novel treatment strategies for neurologic conditions. We have explored whether such an optogenetic approach using the light-driven halorhodopsin chloride pump from Natronomonas pharaonis (NpHR), modified for mammalian CNS expression to hyperpolarize central neurons, may inhibit excessive hyperexcitability and epileptiform activity. We show that a lentiviral vector containing the NpHR gene under the calcium/calmodulin-dependent protein kinase IIα promoter transduces principal cells of the hippocampus and cortex and hyperpolarizes these cells, preventing generation of action potentials and epileptiform activity during optical stimulation. This study proves a principle, that selective hyperpolarization of principal cortical neurons by NpHR is sufficient to curtail paroxysmal activity in transduced neurons and can inhibit stimulation train-induced bursting in hippocampal organotypic slice cultures, which represents a model tissue of pharmacoresistant epilepsy. This study demonstrates that the optogenetic approach may prove useful for controlling epileptiform activity and opens a future perspective to develop it into a strategy to treat epilepsy.

In this Technical Report, Chuong and colleagues introduce Jaws, an archaeon-derived, photoactivatable chloride pump that responds to red light. Owing to its efficiency in absorbing red photons and its large photocurrent, Jaws can be transcranially activated deep in the brain and thus allows noninvasive optogenetic silencing. Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula ( Halobacterium ) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.

Understanding how the brain captures transient experience and converts it into long lasting changes in neural circuits requires the identification and investigation of the specific ensembles of neurons that are responsible for the encoding of each experience. We have developed a Robust Activity Marking (RAM) system that allows for the identification and interrogation of ensembles of neurons. The RAM system provides unprecedented high sensitivity and selectivity through the use of an optimized synthetic activity-regulated promoter that is strongly induced by neuronal activity and a modified Tet-Off system that achieves improved temporal control. Due to its compact design, RAM can be packaged into a single adeno-associated virus (AAV), providing great versatility and ease of use, including application to mice, rats, flies, and potentially many other species. Cre-dependent RAM, CRAM, allows for the study of active ensembles of a specific cell type and anatomical connectivity, further expanding the RAM system’s versatility. Every experience – be it a sight, a sound or a memorable event – activates a unique set of neurons within the brain that together are known as a neuronal ensemble. Identifying these ensembles is key to deciphering how the brain represents experiences and stores them in memory. The most commonly used method for doing so at present relies upon a class of genes called immediate early genes (or IEGs for short). Whenever a neuron becomes active, it switches on its IEGs. By genetically modifying animals to use this mechanism to drive the production of protein markers – such as a fluorescent protein – it is possible to visualize and control the neurons that become activated in response to a stimulus. However, existing IEG-based systems for detecting neuronal activity are not ideal. In particular, these systems could be made more sensitive (so that they are more likely to respond to neuronal activity) and more specific (so that they are more likely to respond only to relevant neuronal activity). Sørensen, Cooper et al. have now developed a new system for tagging recently activated neurons that offers a number of advantages over its predecessors. Known as Robust Activity Marking (RAM), the new system consists of a specially designed DNA sequence that is switched on by neuronal activity. Compared with currently existing systems, the RAM system has low levels of background activity, meaning that it only becomes active in actively firing neurons. It is also extremely sensitive and gives a robust signal. An additional advantage of the RAM system is that the timing of its activation can be precisely controlled. This is useful for identifying those neurons that become active in response to one particular sensory stimulus. The DNA elements in the RAM system that respond to neuronal activity are conserved, which means it could be used in a variety of species, from fruit flies to primates. The relatively small size of the RAM system means that, in contrast to other IEG-based systems, it can be introduced into brains by packaging the entire DNA sequence inside a virus particle that can infect a wide range of experimental species. Finally, the design of the RAM system allows it to be targeted to specific subtypes of neurons and to cells that are connected in particular ways. Together, the multiple advantages of the RAM system over traditional IEG-based systems should make it possible for neuroscientists from many different fields to explore how the brain stores experiences in patterns of neuronal activity.

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

Maladaptive plasticity involving increased expression of AMPA‐type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell‐permeable, high‐affinity (~2 nM) peptide inhibitor, Tat‐P 4 ‐(C5) 2 , of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat‐P 4 ‐(C5) 2 disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA‐receptor surface expression in vivo . Moreover, Tat‐P 4 ‐(C5) 2 administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat‐P 4 ‐(C5) 2 as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non‐tandem protein–protein interaction domains. Synopsis Neuropathic pain is characterized by hypersensitivity to temperature and touch as well as spontaneous outburst of pain. This study identifies a peptide that can inhibit PICK1 and thereby interfere with insertion of excess glutamate receptor underlying the hypersensitivity in neuropathic pain. Tat‐P4‐(C5) 2 is a selective, high‐affinity inhibitor of the PICK1 PDZ domain that effectively interfere with the interaction of the GluA2 subunit of the AMPA type glutamate receptors. The high affinity is achieved in a combination between the Tat peptide and a bivalent PDZ interacting sequence that bring PICK1 into a complex of dimers‐of‐dimers. Tat‐P4‐(C5) 2 disrupt TNFalpha‐induced expression of calcium permeable AMPA receptors and transmission in both DRG neuron and layer 1 and 2 cells in the dorsal horn of the spinal cord. Tat‐P4‐(C5) 2 increases the paw withdrawal threshold to normal in the SNI model of neuropathic pain after intrathecal but not intraplantar injection in accordance with a central mechanism of action. Graphical Abstract Neuropathic pain is characterized by hypersensitivity to temperature and touch as well as spontaneous outburst of pain. This study identifies a peptide that can inhibit PICK1 and thereby interfere with insertion of excess glutamate receptor underlying the hypersensitivity in neuropathic pain.

Disturbances in the brain fluid balance can lead to life‐threatening elevation in intracranial pressure (ICP), which represents a vast clinical challenge. Targeted and efficient pharmaceutical therapy of elevated ICP is not currently available, as the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved. To resolve the quantitative contribution of key choroid plexus transport proteins, this study employs mice with genetic knockout and/or viral choroid plexus‐specific knockdown of aquaporin 1 (AQP1) and the Na+, K+, 2Cl− cotransporter 1 (NKCC1) for in vivo determinations of CSF dynamics, ex vivo choroid plexus for transporter‐mediated clearance of a CSF K+ load, and patient CSF for [K+] quantification. CSF secretion and ICP management occur independently of choroid plexus AQP1 expression, whereas both parameters are reduced by 40% upon choroid plexus NKCC1 knockdown. Elevation of [K+]CSF increases the choroid plexus Na+/K+‐ATPase activity, and favors inwardly‐directed net NKCC1 transport, which, together, promote CSF K+ clearance, while maintaining undisturbed CSF secretion rates. CSF from patients with post‐hemorrhagic hydrocephalus does not display elevated [K+]CSF, suggesting that NKCC1 maintains net outward transport direction during post‐hemorrhagic hydrocephalus formation. Direct or indirect therapeutic modulation of choroid plexus NKCC1 can thus be a potential promising pharmacological approach against brain pathologies associated with elevated ICP. CSF is secreted by the choroid plexus in each ventricle. The choroid plexus water channel, aquaporin 1, is not required for CSF secretion and thus brain fluid dynamics, whereas the Na+, K+, 2Cl‐ cotransporter 1 is a key factor in this process. Elevated ventricular K+ alters the transport activity to clear brain K+ while maintaining the CSF secretion rate.

In the previous decade the first gene therapies were approved in Europe and/or USA, including Glybera® (alipogene tiparvovec) for lipoprotein lipase deficiency (Watanabe et al., 2015), Strimvelis® (ex vivo hematopoietic stem and progenitor cell (HSPC) gene therapy) for adenosine deaminase deficiency-induced severe combined immunodeficiency (ADA-SCID) (Aiuti et al., 2017), Zynteglo® for β-thalassemia (Schuessler-Lenz et al., 2020), Luxturna® (voretigene neparvovec) for inherited retinal dystrophy (Gao et al., 2020), Zolgensma® (onasemnogene abeparvovec) for spinal muscular atrophy (Keeler and Flotte, 2019), and Libmeldy® (ex vivo HSPC gene therapy) for metachromatic leukodystrophy (Bulaklak and Gersbach, 2020). To realize new CNS gene therapies, multiple challenges must be addressed including identifying beneficial therapeutic targets, developing efficient administration and distribution techniques, documenting sustained treatment responses and long-term safety aspects, and demonstrating proof-of-concept for clinical improvements over current available standard of care. O'Carroll et al. provide an overview of the potential and challenges for glial specific gene therapy, since different glial cell types are involved in nervous system pathology, playing roles in neurodegenerative disease and following trauma in the brain and spinal cord (astrocytes, microglia, oligodendrocytes), nerve degeneration and development of pain in peripheral nerves (Schwann cells, satellite cells), retinal diseases (Müller glia), and gut dysbiosis (enteric glia). [...]Tosolini and Sleigh outline how gene therapy can be administered with minimal invasiveness into skeletal muscles for extensive transduction of cells within the spinal cord, brainstem, and sensory ganglia, for treatment of neuronal conditions.

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

Maladaptive plasticity involving increased expression of AMPA‐type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell‐permeable, high‐affinity (~2 nM) peptide inhibitor, Tat‐P4‐(C5)2, of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat‐P4‐(C5)2 disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA‐receptor surface expression in vivo. Moreover, Tat‐P4‐(C5)2 administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat‐P4‐(C5)2 as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non‐tandem protein–protein interaction domains.

Abstract Dopaminergic dysfunction is central to movement disorders and mental diseases. The dopamine transporter (DAT) is essential for the regulation of extracellular dopamine but the genetic and mechanistic link between DAT function and dopamine-related pathologies remains elusive. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here we identify a novel coding DAT variant, DAT-K619N, in a patient with early-onset parkinsonism and comorbid neuropsychiatric disease and in 22 individuals from exome-sequenced samples of neuropsychiatric patients. The variant localizes to the critical C-terminal PDZ-binding motif of DAT and causes reduced uptake capacity, decreased surface expression, and accelerated turnover of DAT in vitro. In vivo, we demonstrate that expression of DAT-K619N in mice and dropsophila imposes impairments in dopamine transmission with accompanying changes in dopamine-directed behaviors. Importantly, both cellular studies and viral overexpression of DAT-K619N in mice show that DAT-K619N has a dominant-negative effect which collectively implies that a single dominant-negative genetic DAT variant can confer risk for neuropsychiatric disease and neurodegenerative early-onset parkinsonism. Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵* On behaf of iPSYCH researchers