Scn1a haploinsufficiency in the prefrontal cortex engages to cognitive impairment and depressive phenotype.

Altered development and function of the prefrontal cortex (PFC) during adolescence is implicated in the origin of mental disorders. Deficits in the GABAergic system prominently contribute to these alterations. Nav1.1 is a voltage-gated Na+ channel critical for normal GABAergic activity. Here, we studied the role of Nav1.1 in PFC function and its potential relationship with the aetiology of mental disorders. Dysfunction of Nav1.1 activity in the medial PFC (mPFC) of adolescent mice enhanced the local excitation/inhibition ratio, resulting in epileptic activity, cognitive deficits and depressive-like behaviour in adulthood, along with a gene expression profile linked to major depressive disorder (MDD). Additionally, it reduced extracellular serotonin concentration in the dorsal raphe nucleus and brain-derived neurotrophic factor expression in the hippocampus, two MDD-related brain areas beyond the PFC. We also observed alterations in oscillatory activity and impaired hippocampal-mPFC coherence during sleep. Finally, we found reduced expression levels of SCN1A, the gene encoding Nav1.1, in post-mortem PFC samples from human MDD subjects. Collectively, our results provide a novel mechanistic framework linking adolescence-specific alterations in Nav1.1 function in the PFC to the pathogenesis of epilepsy and comorbidities such as cognitive impairment and depressive disorders.

Thalamic Foxp2 regulates output connectivity and sensory-motor impairments in a model of Huntington's Disease.

BACKGROUND: Huntington's Disease (HD) is a disorder that affects body movements. Altered glutamatergic innervation of the striatum is a major hallmark of the disease. Approximately 30% of those glutamatergic inputs come from thalamic nuclei. Foxp2 is a transcription factor involved in cell differentiation and reported low in patients with HD. However, the role of the Foxp2 in the thalamus in HD remains unexplored. METHODS: We used two different mouse models of HD, the R6/1 and the HdhQ111 mice, to demonstrate a consistent thalamic Foxp2 reduction in the context of HD. We used in vivo electrophysiological recordings, microdialysis in behaving mice and rabies virus-based monosynaptic tracing to study thalamo-striatal and thalamo-cortical synaptic connectivity in R6/1 mice. Micro-structural synaptic plasticity was also evaluated in the striatum and cortex of R6/1 mice. We over-expressed Foxp2 in the thalamus of R6/1 mice or reduced Foxp2 in the thalamus of wild type mice to evaluate its role in sensory and motor skills deficiencies, as well as thalamo-striatal and thalamo-cortical connectivity in such mouse models. RESULTS: Here, we demonstrate in a HD mouse model a clear and early thalamo-striatal aberrant connectivity associated with a reduction of thalamic Foxp2 levels. Recovering thalamic Foxp2 levels in the mouse rescued motor coordination and sensory skills concomitant with an amelioration of neuropathological features and with a repair of the structural and functional connectivity through a restoration of neurotransmitter release. In addition, reduction of thalamic Foxp2 levels in wild type mice induced HD-like phenotypes. CONCLUSIONS: In conclusion, we show that a novel identified thalamic Foxp2 dysregulation alters basal ganglia circuits implicated in the pathophysiology of HD.

Dual 5-HT3 and 5-HT6 Receptor Antagonist FPPQ Normalizes Phencyclidine-Induced Disruption of Brain Oscillatory Activity in Rats.

Schizophrenia is a severe mental disorder featuring psychotic, depressive, and cognitive alterations. Current antipsychotic drugs preferentially target dopamine D2-R and/or serotonergic 5-HT2A/1A-R. They partly alleviate psychotic symptoms but fail to treat negative symptoms and cognitive deficits. Here we report on the putative antipsychotic activity of (1-[(3-fluorophenyl)sulfonyl]-4-(piperazin-1-yl)-1H-pyrrolo[3,2-c]quinoline dihydrochloride) (FPPQ), a dual serotonin 5-HT3-R/5-HT6-R antagonist endowed with pro-cognitive properties. FPPQ fully reversed phencyclidine-induced decrease of low-frequency oscillations in the medial prefrontal cortex of anaesthetized rats, a fingerprint of antipsychotic activity. This effect was mimicked by the combined administration of the 5-HT3-R and 5-HT6-R antagonists ondansetron and SB-399 885, respectively, but not by either drug alone. In freely moving rats, FPPQ countered phencyclidine-induced hyperlocomotion and augmentation of gamma and high-frequency oscillations in medial prefrontal cortex, dorsal hippocampus, and nucleus accumbens. Overall, this supports that simultaneous blockade of 5-HT3R and 5-HT6-R-like that induced by FPPQ-can be a new target in antipsychotic drug development.

Up and Down γ-Synuclein Transcription in Dopamine Neurons Translates into Changes in Dopamine Neurotransmission and Behavioral Performance in Mice.

The synuclein family consists of α-, ß-, and γ-Synuclein (α-Syn, ß-Syn, and γ-Syn) expressed in the neurons and concentrated in synaptic terminals. While α-Syn is at the center of interest due to its implication in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, limited information exists on the other members. The current study aimed at investigating the biological role of γ-Syn controlling the midbrain dopamine (DA) function. We generated two different mouse models with: (i) γ-Syn overexpression induced by an adeno-associated viral vector and (ii) γ-Syn knockdown induced by a ligand-conjugated antisense oligonucleotide, in order to modify the endogenous γ-Syn transcription levels in midbrain DA neurons. The progressive overexpression of γ-Syn decreased DA neurotransmission in the nigrostriatal and mesocortical pathways. In parallel, mice evoked motor deficits in the rotarod and impaired cognitive performance as assessed by novel object recognition, passive avoidance, and Morris water maze tests. Conversely, acute γ-Syn knockdown selectively in DA neurons facilitated forebrain DA neurotransmission. Importantly, modifications in γ-Syn expression did not induce the loss of DA neurons or changes in α-Syn expression. Collectively, our data strongly suggest that DA release/re-uptake processes in the nigrostriatal and mesocortical pathways are partially dependent on substantia nigra pars compacta /ventral tegmental area (SNc/VTA) γ-Syn transcription levels, and are linked to modulation of DA transporter function, similar to α-Syn.

Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice.

α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson's disease (PD). Point mutations and multiplications of the α-Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α-Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α-Syn (A30P*A53T*α-Syn). We found that A30P*A53T*α-Syn mice at 4-5 months of age showed 3.5-fold increases in human α-Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α-Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND-1233-ASO administration (100 µg/day, 28 days) prevented the α-Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α-Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies.

ER stress in mouse serotonin neurons triggers a depressive phenotype alleviated by ketamine targeting eIF2α signaling.

Depression is a devastating mood disorder that causes significant disability worldwide. Current knowledge of its pathophysiology remains modest and clear biological markers are lacking. Emerging evidence from human and animal models reveals persistent alterations in endoplasmic reticulum (ER) homeostasis, suggesting that ER stress-related signaling pathways may be targets for prevention and treatment. However, the neurobiological basis linking the pathways involved in depression-related ER stress remains unknown. Here, we report that an induced model of ER stress in mouse serotonin (5-HT) neurons is associated with reduced Egr1-dependent 5-HT cellular activity and 5-HT neurotransmission, resulting in neuroplasticity deficits in forebrain regions and a depressive-like phenotype. Ketamine administration engages downstream eIF2α signaling to trigger rapid neuroplasticity events that rescue the depressive-like effects. Collectively, these data identify ER stress in 5-HT neurons as a cellular pathway involved in the pathophysiology of depression and show that eIF2α is critical in eliciting ketamine's fast antidepressant effects.

The Role of α-Synuclein in the Regulation of Serotonin System: Physiological and Pathological Features.

In patients affected by Parkinson's disease (PD), up to 50% of them experience cognitive changes, and psychiatric disturbances, such as anxiety and depression, often precede the onset of motor symptoms and have a negative impact on their quality of life. Pathologically, PD is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc) and the presence of intracellular inclusions, called Lewy bodies and Lewy neurites, composed mostly of α-synuclein (α-Syn). Much of PD research has focused on the role of α-Syn aggregates in the degeneration of SNc DA neurons due to the impact of striatal DA deficits on classical motor phenotypes. However, abundant Lewy pathology is also found in other brain regions including the midbrain raphe nuclei, which may contribute to non-motor symptoms. Indeed, dysfunction of the serotonergic (5-HT) system, which regulates mood and emotional pathways, occurs during the premotor phase of PD. However, little is known about the functional consequences of α-Syn inclusions in this neuronal population other than DA neurons. Here, we provide an overview of the current knowledge of α-Syn and its role in regulating the 5-HT function in health and disease. Understanding the relative contributions to α-Syn-linked alterations in the 5-HT system may provide a basis for identifying PD patients at risk for developing depression and could lead to a more targeted therapeutic approach.

Intracerebral administration of a modified antisense oligonucleotide targeting the dopamine system in a mouse model of Parkinson's disease.

Here, we present an optimized protocol for generating a mouse model overexpressing human α-synuclein in dopamine (DA) neurons driven by an adeno-associated viral (AAV) vector and for the examination of the benefit of an antisense oligonucleotide (ASO)-based therapy on DA neurotransmission under Parkinson's disease (PD)-like conditions. We describe AAV injection, followed by implantation of an osmotic minipump for ASO delivery and a guide cannula for microdialysis to measure DA release. This protocol can be used to evaluate oligonucleotide-based therapies for PD. For complete details on the use and execution of this protocol, please refer to Alarcón-Arís et al. (2020).

Discrimination of motor and sensorimotor effects of phencyclidine and MK-801: Involvement of GluN2C-containing NMDA receptors in psychosis-like models.

Non-competitive NMDA receptor (NMDA-R) antagonists like ketamine, phencyclidine (PCP) and MK-801 are routinely used as pharmacological models of schizophrenia. However, the NMDA-R subtypes, neuronal types (e.g., GABA vs. glutamatergic neurons) and brain regions involved in psychotomimetic actions are not fully understood. PCP activates thalamo-cortical circuits after NMDA-R blockade in reticular thalamic GABAergic neurons. GluN2C subunits are densely expressed in thalamus and cerebellum. Therefore, we examined their involvement in the behavioral and functional effects elicited by PCP and MK-801 using GluN2C knockout (GluN2CKO) and wild-type mice, under the working hypothesis that psychotomimetic effects should be attenuated in mutant mice. PCP and MK-801 induced a disorganized and meandered hyperlocomotion in both genotypes. Interestingly, stereotyped behaviors like circling/rotation, rearings and ataxia signs were dramatically reduced in GluN2CKO mice, indicating a better motor coordination in absence of GluN2C subunits. In contrast, other motor or sensorimotor (pre-pulse inhibition of the startle response) aspects of the behavioral syndrome remained unaltered by GluN2C deletion. PCP and MK-801 evoked a general pattern of c-fos activation in mouse brain (including thalamo-cortical networks) but not in the cerebellum, where they markedly reduced c-fos expression, with significant genotype differences paralleling those in motor coordination. Finally, resting-state fMRI showed an enhanced cortico-thalamic-cerebellar connectivity in GluN2CKO mice, less affected by MK-801 than controls. Hence, the GluN2C subunit allows the dissection of the behavioral alterations induced by PCP and MK-801, showing that some motor effects (in particular, motor incoordination), but not deficits in sensorimotor gating, likely depend on GluN2C-containing NMDA-R blockade in cerebellar circuits.

Human α-synuclein overexpression in mouse serotonin neurons triggers a depressive-like phenotype. Rescue by oligonucleotide therapy.

Anxiety and depression affect 35-50% of patients with Parkinson's disease (PD), often precede the onset of motor symptoms, and have a negative impact on their quality of life. Dysfunction of the serotonergic (5-HT) system, which regulates mood and emotional pathways, occurs during the premotor phase of PD and contributes to a variety of non-motor symptoms. Furthermore, α-synuclein (α-Syn) aggregates were identified in raphe nuclei in the early stages of the disease. However, there are very few animal models of PD-related neuropsychiatric disorders. Here, we develop a new mouse model of α-synucleinopathy in the 5-HT system that mimics prominent histopathological and neuropsychiatric features of human PD. We showed that adeno-associated virus (AAV5)-induced overexpression of wild-type human α-Syn (h-α-Syn) in raphe 5-HT neurons triggers progressive accumulation, phosphorylation, and aggregation of h-α-Syn protein in the 5-HT system. Specifically, AAV5-injected mice displayed axonal impairment in the output brain regions of raphe neurons, and deficits in brain-derived neurotrophic factor (BDNF) expression and 5-HT neurotransmission, resulting in a depressive-like phenotype. Intracerebroventricular treatment with an indatraline-conjugated antisense oligonucleotide (IND-ASO) for four weeks induced an effective and safe reduction of h-α-Syn synthesis in 5-HT neurons and its accumulation in the forebrain, alleviating early deficits of 5-HT function and improving the behavioural phenotype. Altogether, our findings show that α-synucleinopathy in 5-HT neurons negatively affects brain circuits that control mood and emotions, resembling the expression of neuropsychiatric symptoms occurring at the onset of PD. Early preservation of 5-HT function by reducing α-Syn synthesis/accumulation may alleviate PD-related depressive symptoms.

Food craving-like episodes during pregnancy are mediated by accumbal dopaminergic circuits.

Preparation for motherhood requires a myriad of physiological and behavioural adjustments throughout gestation to provide an adequate environment for proper embryonic development1. Cravings for highly palatable foods are highly prevalent during pregnancy2 and contribute to the maintenance and development of gestational overweight or obesity3. However, the neurobiology underlying the distinct ingestive behaviours that result from craving specific foods remain unknown. Here we show that mice, similarly to humans, experience gestational food craving-like episodes. These episodes are associated with a brain connectivity reorganization that affects key components of the dopaminergic mesolimbic circuitry, which drives motivated appetitive behaviours and facilitates the perception of rewarding stimuli. Pregnancy engages a dynamic modulation of dopaminergic signalling through neurons expressing dopamine D2 receptors in the nucleus accumbens, which directly modulate food craving-like events. Importantly, persistent maternal food craving-like behaviour has long-lasting effects on the offspring, particularly in males, leading to glucose intolerance, increased body weight and increased susceptibility to develop eating disorders and anxiety-like behaviours during adulthood. Our results reveal the cognitively motivated nature of pregnancy food cravings and advocates for moderating emotional eating during gestation to prevent deterioration of the offspring's neuropsychological and metabolic health.

Lipid nanoparticles for antisense oligonucleotide gene interference into brain border-associated macrophages.

A colloidal synthesis' proof-of-concept based on the Bligh-Dyer emulsion inversion method was designed for integrating into lipid nanoparticles (LNPs) cell-permeating DNA antisense oligonucleotides (ASOs), also known as GapmeRs (GRs), for mRNA interference. The GR@LNPs were formulated to target brain border-associated macrophages (BAMs) as a central nervous system (CNS) therapy platform for silencing neuroinflammation-related genes. We specifically aim at inhibiting the expression of the gene encoding for lipocalin-type prostaglandin D synthase (L-PGDS), an anti-inflammatory enzyme expressed in BAMs, whose level of expression is altered in neuropsychopathologies such as depression and schizophrenia. The GR@LNPs are expected to demonstrate a bio-orthogonal genetic activity reacting with L-PGDS gene transcripts inside the living system without interfering with other genetic or biochemical circuitries. To facilitate selective BAM phagocytosis and avoid subsidiary absorption by other cells, they were functionalized with a mannosylated lipid as a specific MAN ligand for the mannose receptor presented by the macrophage surface. The GR@LNPs showed a high GR-packing density in a compact multilamellar configuration as structurally characterized by light scattering, zeta potential, and transmission electronic microscopy. As a preliminary biological evaluation of the mannosylated GR@LNP nanovectors into specifically targeted BAMs, we detected in vivo gene interference after brain delivery by intracerebroventricular injection (ICV) in Wistar rats subjected to gene therapy protocol. The results pave the way towards novel gene therapy platforms for advanced treatment of neuroinflammation-related pathologies with ASO@LNP nanovectors.

Involvement of NMDA receptors containing the GluN2C subunit in the psychotomimetic and antidepressant-like effects of ketamine.

Acute ketamine administration evokes rapid and sustained antidepressant effects in treatment-resistant patients. However, ketamine also produces transient perceptual disturbances similarly to those evoked by other non-competitive NMDA-R antagonists like phencyclidine (PCP). Although the brain networks involved in both ketamine actions are not fully understood, PCP and ketamine activate thalamo-cortical networks after NMDA-R blockade in GABAergic neurons of the reticular thalamic nucleus (RtN). Given the involvement of thalamo-cortical networks in processing sensory information, these networks may underlie psychotomimetic action. Since the GluN2C subunit is densely expressed in the thalamus, including the RtN, we examined the dependence of psychotomimetic and antidepressant-like actions of ketamine on the presence of GluN2C subunits, using wild-type and GluN2C knockout (GluN2CKO) mice. Likewise, since few studies have investigated ketamine's effects in females, we used mice of both sexes. GluN2C deletion dramatically reduced stereotyped (circling) behavior induced by ketamine in male and female mice, while the antidepressant-like effect was fully preserved in both genotypes and sexes. Despite ketamine appeared to induce similar effects in both sexes, some neurobiological differences were observed between male and female mice regarding c-fos expression in thalamic nuclei and cerebellum, and glutamate surge in prefrontal cortex. In conclusion, the GluN2C subunit may discriminate between antidepressant-like and psychotomimetic actions of ketamine. Further, the abundant presence of GluN2C subunits in the cerebellum and the improved motor coordination of GluN2CKO mice after ketamine treatment suggest the involvement of cerebellar NMDA-Rs in some behavioral actions of ketamine.

Anti-α-synuclein ASO delivered to monoamine neurons prevents α-synuclein accumulation in a Parkinson's disease-like mouse model and in monkeys.

BACKGROUND: Progressive neuronal death in monoaminergic nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Given that α-synuclein may be an early mediator of the pathological cascade that ultimately leads to neurodegeneration, decreased α-synuclein synthesis will abate neurotoxicity if delivered to the key affected neurons. METHODS: We used a non-viral gene therapy based on a new indatraline-conjugated antisense oligonucleotide (IND-ASO) to disrupt the α-synuclein mRNA transcription selectively in monoamine neurons of a PD-like mouse model and elderly nonhuman primates. Molecular, cell biology, histological, neurochemical and behavioral assays were performed. FINDINGS: Intracerebroventricular and intranasal IND-ASO administration for four weeks in a mouse model with AAV-mediated wild-type human α-synuclein overexpression in dopamine neurons prevented the synthesis and accumulation of α-synuclein in the connected brain regions, improving dopamine neurotransmission. Likewise, the four-week IND-ASO treatment led to decreased levels of endogenous α-synuclein protein in the midbrain monoamine nuclei of nonhuman primates, which are affected early in PD. CONCLUSIONS: The inhibition of α-synuclein production in dopamine neurons and its accumulation in cortical/striatal projection areas may alleviate the early deficits of dopamine function, showing the high translational value of antisense oligonucleotides as a disease modifying therapy for PD and related synucleinopathies. FUNDING: Grants SAF2016-75797-R, RTC-2014-2812-1 and RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; Grant ID 9238, Michael J. Fox Foundation; and Centres for Networked Biomedical Research on Mental Health (CIBERSAM), and on Neurodegenerative Diseases (CIBERNED).