Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-ß), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

Glucocorticoid receptor-dependent therapeutic efficacy of tauroursodeoxycholic acid in preclinical models of spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the ataxin-3 (ATXN3) gene. No effective treatment is available for this disorder, other than symptom-directed approaches. Bile acids have shown therapeutic efficacy in neurodegenerative disease models. Here, we pinpointed tauroursodeoxycholic acid (TUDCA) as an efficient therapeutic, improving the motor and neuropathological phenotype of SCA3 nematode and mouse models. Surprisingly, transcriptomic and functional in vivo data showed that TUDCA acts in neuronal tissue through the glucocorticoid receptor (GR), but independently of its canonical receptor, the farnesoid X receptor (FXR). TUDCA was predicted to bind to the GR, in a similar fashion to corticosteroid molecules. GR levels were decreased in disease-affected brain regions, likely due to increased protein degradation as a consequence of ATXN3 dysfunction being restored by TUDCA treatment. Analysis of a SCA3 clinical cohort showed intriguing correlations between the peripheral expression of GR and the predicted age at disease onset in presymptomatic subjects and FKBP5 expression with disease progression, suggesting this pathway as a potential source of biomarkers for future study. We have established a novel in vivo mechanism for the neuroprotective effects of TUDCA in SCA3 and propose this readily available drug for clinical trials in SCA3 patients.

Treating Parkinson's Disease with Human Bone Marrow Mesenchymal Stem Cell Secretome: A Translational Investigation Using Human Brain Organoids and Different Routes of In Vivo Administration.

Parkinson's disease (PD) is the most common movement disorder, characterized by the progressive loss of dopaminergic neurons from the nigrostriatal system. Currently, there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Mesenchymal stem cells (MSCs) are one of the most extensively studied cell sources for regenerative medicine applications, particularly due to the release of soluble factors and vesicles, known as secretome. The main goal of this work was to address the therapeutic potential of the secretome collected from bone-marrow-derived MSCs (BM-MSCs) using different models of the disease. Firstly, we took advantage of an optimized human midbrain-specific organoid system to model PD in vitro using a neurotoxin-induced model through 6-hydroxydopamine (6-OHDA) exposure. In vivo, we evaluated the effects of BM-MSC secretome comparing two different routes of secretome administration: intracerebral injections (a two-site single administration) against multiple systemic administration. The secretome of BM-MSCs was able to protect from dopaminergic neuronal loss, these effects being more evident in vivo. The BM-MSC secretome led to motor function recovery and dopaminergic loss protection; however, multiple systemic administrations resulted in larger therapeutic effects, making this result extremely relevant for potential future clinical applications.

Mitochondrial Dysfunction and Decreased Cytochrome c in Cell and Animal Models of Machado-Joseph Disease.

Mitochondrial dysfunction has been described in many neurodegenerative disorders; however, there is less information regarding mitochondrial deficits in Machado-Joseph disease (MJD), a polyglutamine (polyQ) disorder caused by CAG repeat expansion in the ATXN3 gene. In the present study, we characterized the changes in mitochondrial function and biogenesis markers in two MJD models, CMVMJD135 (MJD135) transgenic mice at a fully established phenotype stage and tetracycline-regulated PC6-3 Q108 cell line expressing mutant ataxin-3 (mATXN3). We detected mATXN3 in the mitochondrial fractions of PC6-3 Q108 cells, suggesting the interaction of expanded ATXN3 with the organelle. Interestingly, in both the cerebella of the MJD135 mouse model and in PC6-3 Q108 cells, we found decreased mitochondrial respiration, ATP production and mitochondrial membrane potential, strongly suggesting mitochondrial dysfunction in MJD. Also, in PC6-3 Q108 cells, an additional enhanced glycolytic flux was observed. Supporting the functional deficits observed in MJD mitochondria, MJD135 mouse cerebellum and PC6-3 Q108 cells showed reduced cytochrome c mRNA and protein levels. Overall, our findings show compromised mitochondrial function associated with decreased cytochrome c levels in both cell and animal models of MJD.

Genetic Ablation of Inositol 1,4,5-Trisphosphate Receptor Type 2 (IP3R2) Fails to Modify Disease Progression in a Mouse Model of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.

Cell-based therapeutic strategies for treatment of spinocerebellar ataxias: an update.

Spinocerebellar ataxias are heritable neurodegenerative diseases caused by a cytosine-adenine-guanine expansion, which encodes a long glutamine tract (polyglutamine) in the respective wild-type protein causing misfolding and protein aggregation. Clinical features of polyglutamine spinocerebellar ataxias include neuronal aggregation, mitochondrial dysfunction, decreased proteasomal activity, and autophagy impairment. Mutant polyglutamine protein aggregates accumulate within neurons and cause neural dysfunction and death in specific regions of the central nervous system. Spinocerebellar ataxias are mostly characterized by progressive ataxia, speech and swallowing problems, loss of coordination and gait deficits. Over the past decade, efforts have been made to ameliorate disease symptoms in patients, yet no cure is available. Previous studies have been proposing the use of stem cells as promising tools for central nervous system tissue regeneration. So far, pre-clinical trials have shown improvement in various models of neurodegenerative diseases following stem cell transplantation, including animal models of spinocerebellar ataxia types 1, 2, and 3. However, contrasting results can be found in the literature, depending on the animal model, cell type, and route of administration used. Nonetheless, clinical trials using cellular implants into degenerated brain regions have already been applied, with the expectation that these cells would be able to differentiate into the specific neuronal subtypes and re-populate these regions, reconstructing the affected neural network. Meanwhile, the question of how feasible it is to continue such treatments remains unanswered, with long-lasting effects being still unknown. To establish the value of these advanced therapeutic tools, it is important to predict the actions of the transplanted cells as well as to understand which cell type can induce the best outcomes for each disease. Further studies are needed to determine the best route of administration, without neglecting the possible risks of repetitive transplantation that these approaches so far appear to demand. Despite the challenges ahead of us, cell-transplantation therapies are reported to have transient but beneficial outcomes in spinocerebellar ataxias, which encourages efforts towards their improvement in the future.

Intrathecal Injection of the Secretome from ALS Motor Neurons Regulated for miR-124 Expression Prevents Disease Outcomes in SOD1-G93A Mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with short life expectancy and no effective therapy. We previously identified upregulated miR-124 in NSC-34-motor neurons (MNs) expressing human SOD1-G93A (mSOD1) and established its implication in mSOD1 MN degeneration and glial cell activation. When anti-miR-124-treated mSOD1 MN (preconditioned) secretome was incubated in spinal cord organotypic cultures from symptomatic mSOD1 mice, the dysregulated homeostatic balance was circumvented. To decipher the therapeutic potential of such preconditioned secretome, we intrathecally injected it in mSOD1 mice at the early stage of the disease (12-week-old). Preconditioned secretome prevented motor impairment and was effective in counteracting muscle atrophy, glial reactivity/dysfunction, and the neurodegeneration of the symptomatic mSOD1 mice. Deficits in corticospinal function and gait abnormalities were precluded, and the loss of gastrocnemius muscle fiber area was avoided. At the molecular level, the preconditioned secretome enhanced NeuN mRNA/protein expression levels and the PSD-95/TREM2/IL-10/arginase 1/MBP/PLP genes, thus avoiding the neuronal/glial cell dysregulation that characterizes ALS mice. It also prevented upregulated GFAP/Cx43/S100B/vimentin and inflammatory-associated miRNAs, specifically miR-146a/miR-155/miR-21, which are displayed by symptomatic animals. Collectively, our study highlights the intrathecal administration of the secretome from anti-miR-124-treated mSOD1 MNs as a therapeutic strategy for halting/delaying disease progression in an ALS mouse model.

Microglial Depletion Has No Impact on Disease Progression in a Mouse Model of Machado-Joseph Disease.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder (ND). While most research in NDs has been following a neuron-centric point of view, microglia are now recognized as crucial in the brain. Previous work revealed alterations that point to an increased activation state of microglia in the brain of CMVMJD135 mice, a MJD mouse model that replicates the motor symptoms and neuropathology of the human condition. Here, we investigated the extent to which microglia are actively contributing to MJD pathogenesis and symptom progression. For this, we used PLX3397 to reduce the number of microglia in the brain of CMVMJD135 mice. In addition, a set of statistical and machine learning models were further implemented to analyze the impact of PLX3397 on the morphology of the surviving microglia. Then, a battery of behavioral tests was used to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Although PLX3397 treatment substantially reduced microglia density in the affected brain regions, it did not affect the motor deficits seen in CMVMJD135 mice. In addition to reducing the number of microglia, the treatment with PLX3397 induced morphological changes suggestive of activation in the surviving microglia, the microglia of wild-type animals becoming similar to those of CMVMJD135 animals. These results suggest that microglial cells are not key contributors for MJD progression. Furthermore, the impact of PLX3397 on microglial activation should be taken into account in the interpretation of findings of ND modification seen upon treatment with this CSF1R inhibitor.

Cerebellar neuronal dysfunction accompanies early motor symptoms in spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is an adult-onset, progressive ataxia. SCA3 presents with ataxia before any gross neuropathology. A feature of many cerebellar ataxias is aberrant cerebellar output that contributes to motor dysfunction. We examined whether abnormal cerebellar output was present in the CMVMJD135 SCA3 mouse model and, if so, whether it correlated with the disease onset and progression. In vivo recordings showed that the activity of deep cerebellar nuclei neurons, the main output of the cerebellum, was altered. The aberrant activity correlated with the onset of ataxia. However, although the severity of ataxia increased with age, the severity of the aberrant cerebellar output was not progressive. The abnormal cerebellar output, however, was accompanied by non-progressive abnormal activity of their upstream synaptic inputs, the Purkinje cells. In vitro recordings indicated that alterations in intrinsic Purkinje cell pacemaking and in their synaptic inputs contributed to abnormal Purkinje cell activity. These findings implicate abnormal cerebellar physiology as an early, consistent contributor to pathophysiology in SCA3, and suggest that the aberrant cerebellar output could be an appropriate therapeutic target in SCA3.

Profiling Microglia in a Mouse Model of Machado-Joseph Disease.

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado-Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.

Preclinical Assessment of Mesenchymal-Stem-Cell-Based Therapies in Spinocerebellar Ataxia Type 3.

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)-or Machado-Joseph disease (MJD)-is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation-particularly in the cerebellum and basal ganglia-while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.

Unilateral Intrastriatal 6-Hydroxydopamine Lesion in Mice: A Closer Look into Non-Motor Phenotype and Glial Response.

Parkinson's disease (PD) is a prevalent movement disorder characterized by the progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). The 6-hydroxydopamine (6-OHDA) lesion is still one of the most widely used techniques for modeling Parkinson's disease (PD) in rodents. Despite commonly used in rats, it can be challenging to reproduce a similar lesion in mice. Moreover, there is a lack of characterization of the extent of behavioral deficits and of the neuronal loss/neurotransmitter system in unilateral lesion mouse models. In this study, we present an extensive behavioral and histological characterization of a unilateral intrastriatal 6-OHDA mouse model. Our results indicate significant alterations in balance and fine motor coordination, voluntary locomotion, and in the asymmetry's degree of forelimb use in 6-OHDA lesioned animals, accompanied by a decrease in self-care and motivational behavior, common features of depressive-like symptomatology. These results were accompanied by a decrease in tyrosine hydroxylase (TH)-labelling and dopamine levels within the nigrostriatal pathway. Additionally, we also identify a marked astrocytic reaction, as well as proliferative and reactive microglia in lesioned areas. These results confirm the use of unilateral intrastriatal 6-OHDA mice for the generation of a mild model of nigrostriatal degeneration and further evidences the recapitulation of key aspects of PD, thereby being suitable for future studies beholding new therapeutical interventions for this disease.

IP3 R2 null mice display a normal acquisition of somatic and neurological development milestones.

Astrocytes are key players in the regulation of brain development and function. They sense and respond to the surrounding activity by elevating their intracellular calcium (Ca2+ ) levels. These astrocytic Ca2+ elevations emerge from different sources and display complex spatio-temporal properties. Ca2+ elevations are spatially distributed in global (soma and main processes) and/or focal regions (microdomains). The inositol 1,4,5-trisphosphate receptor type 2 knockout (IP3 R2 KO) mouse model lacks global Ca2+ elevations in astrocytes, and it has been used by different laboratories. However, the constitutive deletion of IP3 R2 during development may trigger compensating phenotypes, which could bias the results of experiments using developing or adult mice. To address this issue, we performed a detailed neurodevelopmental evaluation of male and female IP3 R2 KO mice, during the first 21 days of life, as well as an evaluation of motor function, strength and neurological reflexes in adult mice. Our results show that male and female IP3 R2 KO mice display a normal acquisition of developmental milestones, as compared with wild-type (WT) mice. We also show that IP3 R2 KO mice display normal motor coordination, strength and neurological reflexes in adulthood. To exclude a potential compensatory overexpression of other IP3 Rs, we quantified the relative mRNA levels of all 3 subtypes, in brain tissue. We found that, along with the complete deletion of Itpr2, there is no compensatory expression of Itpr1 or Itrp3. Overall, our results show that the IP3 R2 KO mouse is a reliable model to study the functional impact of global IP3 R2-dependent astrocytic Ca2+ elevations.

Splenic sympathetic signaling contributes to acute neutrophil infiltration of the injured spinal cord.

BACKGROUND: Alterations in the immune system are a complication of spinal cord injury (SCI) and have been linked to an excessive sympathetic outflow to lymphoid organs. Still unknown is whether these peripheral immune changes also contribute for the deleterious inflammatory response mounted at the injured spinal cord. METHODS: We analyzed different molecular outputs of the splenic sympathetic signaling for the first 24 h after a thoracic compression SCI. We also analyzed the effect of ablating the splenic sympathetic signaling to the innate immune and inflammatory response at the spleen and spinal cord 24 h after injury. RESULTS: We found that norepinephrine (NE) levels were already raised at this time-point. Low doses of NE stimulation of splenocytes in vitro mainly affected the neutrophils' population promoting an increase in both frequency and numbers. Interestingly, the interruption of the sympathetic communication to the spleen, by ablating the splenic nerve, resulted in reduced frequencies and numbers of neutrophils both at the spleen and spinal cord 1 day post-injury. CONCLUSION: Collectively, our data demonstrates that the splenic sympathetic signaling is involved in the infiltration of neutrophils after spinal cord injury. Our findings give new mechanistic insights into the dysfunctional regulation of the inflammatory response mounted at the injured spinal cord.

Polyglutamine spinocerebellar ataxias: emerging therapeutic targets.

INTRODUCTION: Six of the most frequent dominantly inherited spinocerebellar ataxias (SCAs) worldwide - SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17 - are caused by an expansion of a polyglutamine (polyQ) tract in the corresponding proteins. While the identification of the causative mutation has advanced knowledge on the pathogenesis of polyQ SCAs, effective therapeutics able to mitigate the severe clinical manifestation of these highly incapacitating disorders are not yet available. AREAS COVERED: This review provides a comprehensive and critical perspective on well-established and emerging therapeutic targets for polyQ SCAs; it aims to inspire prospective drug discovery efforts. EXPERT OPINION: The landscape of polyQ SCAs therapeutic targets and strategies includes (1) the mutant genes and proteins themselves, (2) enhancement of endogenous protein quality control responses, (3) abnormal protein-protein interactions of the mutant proteins, (4) disturbed neuronal function, (5) mitochondrial function, energy availability and oxidative stress, and (6) glial dysfunction, growth factor or hormone imbalances. Challenges include gaining a clearer definition of therapeutic targets for the drugs in clinical development, the discovery of novel drug-like molecules for challenging key targets, and the attainment of a stronger translation of preclinical findings to the clinic.

Deficiency in classical nonhomologous end-joining-mediated repair of transcribed genes is linked to SCA3 pathogenesis.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.

Neurotoxic effects of MPTP on mouse cerebral cortex: Modulation of neuroinflammation as a neuroprotective strategy.

Parkinson's disease (PD) is a progressive neurological disorder, mainly characterized by the progressive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) and by the presence of intracellular inclusions, known as Lewy bodies. Despite SNpc being considered the primary affected region in PD, the neuropathological features are confined solely to the nigro-striatal axis. With disease progression other brain regions are also affected, namely the cerebral cortex, although the spreading of the neurologic damage to this region is still not completely unraveled. Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid that has been shown to have antioxidant properties and to exhibit a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice model of PD. Moreover, TUDCA anti-inflammatory properties have been reported in glial cells, making it a prominent therapeutic agent in PD. Here, we used C57BL/6 mice injected with MPTP in a sub-acute paradigm aiming to investigate if the neurotoxic effects of MPTP could be extended to the cerebral cortex. In parallel, we evaluated the anti-oxidant, neuroprotective and anti-inflammatory effects of TUDCA. The anti-inflammatory mechanisms elicited by TUDCA were further dissected in microglia cells. Our results show that MPTP leads to a decrease of ATP and activated AMP-activated protein kinase levels in mice cortex, and to a transient increase in the expression of antioxidant downstream targets of nuclear factor erythroid 2 related factor 2 (Nrf-2), and parkin. Notably, MPTP increases pro-inflammatory markers, while down-regulating the expression of the anti-inflammatory protein Annexin-A1 (ANXA1). Importantly, we show that TUDCA treatment prevents the deleterious effects of MPTP, sustains increased levels of antioxidant enzymes and parkin, and most of all negatively modulates neuroinflammation and up-regulates ANXA1 expression. Additionally, results from cellular models using microglia corroborate TUDCA modulation of ANXA1 synthesis, linking inhibition of neuroinflammation and neuroprotection by TUDCA.

Citalopram Reduces Aggregation of ATXN3 in a YAC Transgenic Mouse Model of Machado-Joseph Disease.

Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is a fatal polyglutamine disease with no disease-modifying treatment. The selective serotonin reuptake inhibitor citalopram was shown in nematode and mouse models to be a compelling repurposing candidate for Machado-Joseph disease therapeutics. We sought to confirm the efficacy of citalopram to decrease ATXN3 aggregation in an unrelated mouse model of Machado-Joseph disease. Four-week-old YACMJD84.2 mice and non-transgenic littermates were given citalopram 8 mg/kg in drinking water or water for 10 weeks. At the end of treatment, brains were collected for biochemical and pathological analyses. Brains of citalopram-treated YACMJD84.2 mice showed an approximate 50% decrease in the percentage of cells containing ATXN3-positive inclusions in the substantia nigra and three examined brainstem nuclei compared to controls. No differences in ATXN3 inclusion load were observed in deep cerebellar nuclei of mice. Citalopram effect on ATXN3 aggregate burden was corroborated by immunoblotting analysis. While lysates from the brainstem and cervical spinal cord of citalopram-treated mice showed a decrease in all soluble forms of ATXN3 and a trend toward reduction of insoluble ATXN3, no differences in ATXN3 levels were found between cerebella of citalopram-treated and vehicle-treated mice. Citalopram treatment altered levels of select components of the cellular protein homeostatic machinery that may be expected to enhance the capacity to refold and/or degrade mutant ATXN3. The results here obtained in a second independent mouse model of Machado-Joseph disease further support citalopram as a potential drug to be repurposed for this fatal disorder.

Preclinical Evidence Supporting Early Initiation of Citalopram Treatment in Machado-Joseph Disease.

Spinocerebellar ataxias are dominantly inherited neurodegenerative disorders with no disease-modifying treatment. We previously identified the selective serotonin reuptake inhibitor citalopram as a safe and effective drug to be repurposed for Machado-Joseph disease. Pre-symptomatic treatment of transgenic (CMVMJD135) mice strikingly ameliorated mutant ataxin-3 (ATXN3) pathogenesis. Here, we asked whether citalopram treatment initiated at a post-symptomatic age would still show efficacy. We used a cohort of CMVMJD135 mice that shows increased phenotypic severity and faster disease progression (CMVMJD135hi) compared to the mice used in the first trial. Groups of hemizygous CMVMJD135hi mice were orally treated with citalopram. Behavior, protein analysis, and pathology assessment were performed blindly to treatment. Our results show that even when initiated after symptom onset, treatment of CMVMJD135hi mice with citalopram ameliorated motor coordination and balance, attenuating disease progression, albeit to a lesser extent than that seen with pre-symptomatic treatment initiation. There was no impact on ATXN3 aggregation, which contrasts with the robust reduction in ATXN3-positive inclusions observed in CMVMJD135 mice, when treated pre-symptomatically. Post-symptomatic treatment of CMVMJD135hi mice revealed, however, a limited neuroprotective effect by showing a tendency to repair cerebellar calbindin staining, and to increase the number of motor neurons and of NeuN-positive cells in certain brain regions. While supporting that early initiation of treatment with citalopram leads to a marked increase in efficacy, these results strengthen our previous observation that modulation of serotonergic signaling by citalopram is a promising therapeutic approach for Machado-Joseph disease even after symptom onset.