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.We thank to the Coordenação de Aperfeiçoamento de Nivel Superior (CAPES-PDSE: 19/2016 88881.135527/2016-01), Brazil, for their financial support via a scholarship awarded to C.C.-S. We also thank JAE-Intro Program, CSIC (JAEINT_20_02116), Spain, for their financial support via a scholarship awarded to M.T.-L. This study was supported by grants SAF2016-75797-R, PID2019-105136RB-100, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; and CB/07/09/0034 Center for Networked Biomedical Research on Mental Health (CIBERSAM)

Age-dependent multisystem parkinsonian features in a novel neuromelanin-producing transgenic mouse model

Trabajo presentado en el 19th National Meeting of the Spanish Society of Neuroscience, celebrado en Lleida (España), del 3 al 5 de noviembre de 2021Parkinson’s disease (PD) is characterized by a preferential degeneration of neurons that accumulate with age the pigment neuromelanin, especially neurons from substantia nigra (SN) and locus coeruleus (LC). We aim to characterize the consequences of age-dependent intracellular neuromelanin accumulation in catecholaminergic neuronal populations to understand the relationship between this process and the vulnerability of these cells in PD, as well as its impact on healthy brain aging. We previously generated a rat model exhibiting progressive unilateral SN production of neuromelanin that showed parkinsonian-like neuropathology and motor deficits1. Here, we generated a new neuromelanin-producing rodent model, based on the tissue-specific constitutive expression of human tyrosinase (hTyr) under the tyrosine hydroxylase (TH) promoter (Tg-TH-hTyr), that mimics the bilateral distribution of pigmentation within the aging human brain (i.e. catecholaminergic groups A1-A142). In parallel to neuromelanin intracellular buildup, Tg-TH-hTyr mice exhibited major PD features, including motor and non-motor behavioral alterations, inclusion body formation and degeneration of specific catecholaminergic neuronal groups. Genome-wide transcriptomic analysis of neuromelanin-laden neurons revealed alterations in PD-related biological pathways that correlate with human PD postmortem studies. Our results show that modelling human neuromelanin accumulation in rodents leads to age-dependent catecholaminergic dysfunction and molecular alterations resulting in motor and non-motor deficits, which is relevant to PD pathology and brain aging.Peer reviewe

Transcriptomic changes linked to age-dependent neuromelanin accumulation in a new Parkinson's disease mouse model

Resumen del trabajo presentado en el 50th Annual Meeting Society for Neuroscience, celebrado de forma virtual del 8 al 11 de noviembre de 2021In Parkinson's disease (PD) there is a preferential degeneration of neuromelanin (NM)-containing neurons, especially neurons from the Substantia Nigra (SN) but also from the Ventral Tegmental Area (VTA) and Locus Coeruleus (LC). We generated a new NM-producing mouse model, based on the tissue-specific constitutive expression of human tyrosinase (hTyr) under the tyrosine hydroxylase (TH) promoter (tgNM), that mimics the distribution and age-dependent accumulation of NM in the human brain (i.e. catecholaminergic groups A1-A14). TgNM mice exhibited major PD features, including both motor and non-motor behavioral alterations, inclusion body formation, neuronal degeneration in lower brainstem areas (LC) together with neuronal dysfunction in higher brainstem areas (SN and VTA). In order to understand the mechanisms by which NM accumulation in specific brain areas ultimately interferes with the normal functioning of cells, we characterized genome-wide transcriptomic changes linked to the intracellular presence and progressive accumulation of NM in two NM-accumulating neuronal subpopulations (SN and VTA) that are known to be differentially susceptible to PD pathology. We selectively isolated single dopaminergic NM-containing neurons by laser capture microdissection from male and female wild-type and tgNM animals at 3 months, 12 months and 20 months of age (n=4-6 mice per group). We performed differential expression analysis, resulting in statistically significant differentially expressed genes at all ages (p-value<0.5). Gene-set enrichment analysis (GSEA) with Reactome Pathway Database led to the identification of altered biological pathways in tgNM related to neuroinflammation, vesicle-mediated transport and lipid metabolism, transcription and translation, mitochondrial function and cell cycle (senescence) (False Discovery Rate<0.05). Targeted-based validation of candidate RNA species was performed in microdissected samples by quantitative real-time PCR and candidate biological pathways were validated at the protein level by western blot in dissected ventral midbrain tissues from biological replicates. The transcriptomic profiles identified in this project contribute to our understanding of selective vulnerability in PD and brain aging, and points to key biological pathways and molecular targets in prodromal and early PD

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.This study was supported by grants SAF2016-75797-R, PID2019-105136RB-100, Retos- Colaboración Subprogram RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; and CB/07/09/0034 Center for Networked Biomedical Research on Mental Health (CIBERSAM)

Emotional-like disorders in a mouse model overexpressing human ¿-synuclein in serotonin neurons: Focus on synaptic density markers

Trabajo presentado en el VIII Laboratorio de ideas para jóvenes investigadores CIBERSAM: Impacto de la pandemia COVID-19, celebrado de forma virtual del 25 al 27 de mayo de 2021Anxiety and depression are the most prevalent neuropsychiatric disorders in Parkinson’s disease ( population 51 and 41 respectively), often preceding the appearance of motor symptoms, and reducing the health related quality of life However, mechanisms of anxiety/depression in PD are not known in detail Degeneration of serotonergic 5 HT) neurons, which regulate mood and emotional pathways, occurs during the PD prodromal phase and contributes to a variety of non motor symptoms Besides, recent neuroimaging studies suggest that in the early phases of PD, synaptic and axonal damage anticipate the onset of a frank neuronal death Paralleling, even post mortem studies on the brain of affected patients and animal models support that synapses might represent the primary sites of functional and pathological changes In fact, previous studies of our group showed that the overexpression of human α synuclein in the mouse 5 HT neurons induced by the AAV 5 vector leads to increased axonal transport of human α synuclein and the appearance of axonal swellings in anatomically connected brain areas before cell death occurs, along with a depressive phenotype Alarcón Arís et al 2020 Miquel Rio et al, in preparation)Peer reviewe

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).This study was supported by grants SAF2016-75797-R and PID2019-105136RB-100, Ministry of Science and Innovation (MICINN) and European Regional Development Fund (ERDF), EU, and CB/07/09/0034 Center for Networked Biomedical Research on Mental Health (CIBERSAM)

Linking endoplasmic reticulum stress to depression: involvement of eIF2¿ pathway

Trabajo presentado en el ECNP Workshop for Early Career Scientists in Europe, celebrado en Niza (Francia) del 16 al 19 de marzo de 202

Antidepressant actions of ketamine engage cellular mechanisms of endoplasmic reticulum stress by the eIF2¿ pathway

Trabajo presentado en el 19th National Meeting of the Spanish Society of Neuroscience, celebrado en Lleida (España), del 3 al 5 de noviembre de 2021Chaperone proteins and folding enzymes in endoplasmic reticulum (ER) perform a key role in proteostasis, which can be disrupted by numerous factors leading to an accumulation of unfolded proteins in the lumen. It results in ER stress and unfolded protein response (UPR) is elicited to restore homeostasis. However, under prolonged cellular stress, the UPR pathway can lead to cell dysfunction/loss. Impaired ER mechanisms are responsible for neurodegeneration in numerous human diseases and there is also growing evidence that ER stress is implicated in the neuronal dysfunctions of neuropsychiatric disorders. The present study was aimed to check the hypothesis that ER stress and UPR pathway over-activation in the serotonin (5-HT) neurons are involved in the cellular pathological mechanisms of anxiety and depression by causing an impaired proteasome function. ER stress was induced by a single local application of tunicamycin (200 μg/μl) in dorsal raphe nucleus of mice. We examined tunicamycin effects on proteins related to ER stress, UPR, and apoptosis, on serotonin function as well as on anxiety- and depression-like behaviors. Tunicamycin rapidly induced ER stress in 5-HT neurons, leading to a time-dependent increase in GRP78 protein levels. Furthermore, CHOP protein, which triggers apoptosis pathways, was also increased 7 days after tunicamycin infusion. ER stress led to an increased eIF2α and eEF2 phosphorylation, suggesting the activation of PERK pathway in 5-HT neurons. Tunicamycin-treated mice exhibited an anxious-depressive phenotype and showed altered 5-HT neurotransmission in medial prefrontal cortex. A single dose of ketamine (10 mg/kg, ip) reversed the depressive phenotype 30 minutes post-administration, which is linked to reduced levels of phosphorylated eIF2α and recovery of proteostasis. The results strongly indicate that ER stress and UPR may represent cellular pathogenic mechanisms in the development of mood disorders and the eIF2α pathway is central for antidepressant activity of ketamine.SAF2016-75797-R, PID2019-105136RB-100 (MINECO-ERDF)Peer reviewe

Antidepressant actions of ketamine engage cellular mechanisms of endoplasmic reticulum stress by the eIF2¿ pathway

Trabajo presentado en FENS 2022, celebrado en París (Francia), del 9 al 13 de julio de 2022Aims: Depression is a devastating mood disorder that causes profound disability worldwide. Despite the growing number of antidepressant medications available, treatment options for depression are limited. Therefore, it is imperative to understand the etiology and pathophysiology of depression to discover novel therapeutic targets of action. Here, we explore how endoplasmic reticulum (ER) stress might play an important role in the pathophysiology of depression and how the antidepressant ketamine actions involve ER pathways Methods: We generated a mouse model of ER stress in serotonin (5-HT) neurons using the stressor tunicamycin (200 μg/μl). We examined ER/UPR pathway markers by Western blot, neuroplasticity gene expression (BDNF, TrkB, VEGF, Neuritin, PSD95, and Zif268) by in situ hybridization, 5-HT release by microdialysis, and behavioral depressive-like phenotype. Ketamine (10 mg/kg, i.p.) was used to reverse the ER stress-induced depressive mouse model. Results: Tunicamycin-induced ER stress in 5-HT neurons left a time-dependent increase in GRP78 and CHOP protein levels. In addition, increased phosphorylation of eIF2α and eEF2 was found, suggesting activation of PERK pathway. Tunicamycin-treated mice exhibited an anxious/depressive phenotype, reduced 5-HT release in the medial prefrontal cortex, and changes in neuroplasticity gene expression in 5-HT projection areas. A single dose of ketamine reversed the depressive phenotype 30 minutes later, which is associated with reduced levels of phosphorylated eIF2α and recovery of BDNF expression. Conclusions: The results strongly indicate that ER stress and UPR may represent cellular pathogenic mechanisms in the development of mood disorders and that eIF2α pathway is central for the antidepressant activity of ketamine

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.This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2015-68346-P and PI16/00287) co-financed by European Regional Development Fund; Generalitat de Catalunya (CERCA Program; 2014-SGR798; 2017-SGR717; 2016FI-B00285 to M.T.-G., co-financed by the European Social Fund); Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM)