Striatal Cultures Research Articles

Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human exvivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.

NECAB2 is an endosomal protein important for striatal function

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

Striatal mGlu5-mediated synaptic plasticity is independently regulated by location-specific receptor pools and divergent signaling pathways

Metabotropic glutamate receptor 5 (mGlu5) is widely expressed throughout the central nervous system and is involved in neuronal function, synaptic transmission, and a number of neuropsychiatric disorders such as depression, anxiety, and autism. Recent work from this lab showed that mGlu5 is one of a growing number of G protein-coupled receptors that can signal from intracellular membranes where it drives unique signaling pathways, including upregulation of extracellular signal-regulated kinase (ERK1/2), ETS transcription factor Elk-1, and activity-regulated cytoskeleton-associated protein (Arc). To determine the roles of cell surface mGlu5 as well as the intracellular receptor in a well-known mGlu5 synaptic plasticity model such as long-term depression, we used pharmacological isolation and genetic and physiological approaches to analyze spatially restricted pools of mGlu5 in striatal cultures and slice preparations. Here we show that both intracellular and cell surface receptors activate the phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, whereas only intracellular mGlu5 activates protein phosphatase 2 and leads to fragile X mental retardation protein degradation and de novo protein synthesis followed by a protein synthesis-dependent increase in Arc and post-synaptic density protein 95. However, both cell surface and intracellular mGlu5 activation lead to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA2 internalization and chemically induced long-term depression albeit via different signaling mechanisms. These data underscore the importance of intracellular mGlu5 in the cascade of events associated with sustained synaptic transmission in the striatum.

Effect of calcitriol and calcium on basal ganglia calcification in hypoparathyroidism: experimental models.

Basal ganglia calcification (BGC) is a common complication in hypoparathyroid patients, linked to hyperphosphatemia and altered vitamin-D and calcium homeostasis following conventional therapy. The pathogenesis of BGC in hypoparathyroidism is not clear. Recently, we developed an ex vivo model of BGC using rat-striatal cell culture in 10.0 mmol/L of β-glycerophosphate (31.8 mg/dL phosphate). However, the effect of 1,25(OH)2 D, calcium, and milder phosphate excess on BGC in hypoparathyroidism is not known. This study describes two modified ex vivo models investigating pathogenesis of BGC in 'drug-naïve' and 'conventionally treated' hypoparathyroid state. The first modification involved striatal cells cultured in low concentration 1,25(OH)2D (16.0 pg/mL), ionized calcium(0.99 mmol/L), hPTH(1-34) (6.0 pg/mL), and 2.68 mmol/L (8.3 mg/dL) of phosphate akin to 'drug-naïve' state for 24 days. In second modification, striatal cells were exposed to 46.0 pg/mL of 1,25(OH)2D, normal ionized calcium of 1.17 mmol/L, and 2.20 mmol/L (6.8 mg/dL) of phosphate akin to 'conventionally treated' state. Striatal cell culture under 'drug-naïve' state showed that even 16.0 pg/mL of 1,25(OH)2D enhanced the calcification. In 'conventionally treated' model, striatal cell calcification was enhanced in 54% cases over 'drug-naïve' state. Calcification in 'conventionally treated' state further increased on increasing phosphate to 8.3 mg/dL, suggesting importance of phosphatemic control in hypoparathyroid patients. Striatal cells in 'drug-naïve' state showed increased mRNA expression of pro-osteogenic Wnt3a, Cd133,Vglut-1-neuronal phosphate-transporters, calcium-ion channel-Trvp2,Alp, and Collagen-1α and decreased expression of Ca-II. These models suggest that in 'drug-naïve' state, 1,25(OH)2D along with moderately elevated phosphate increases the expression of pro-osteogenic molecules to induce BGC. Although normalization of calcium in 'conventionally treated' state increased the expression of Opg, Osterix, Alp, and Cav2, calcification increased only in a subset, akin to variation in progression of BGC in hypoparathyroid patients on conventional therapy.

Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia.

HIV/HIV-1 Tat and morphine independently increase pathologic phosphorylation of TAR DNA binding protein 43 in the striatum. HIV- and opioid-induced pathologic phosphorylation of TAR DNA binding protein 43 may involve enhanced CK2 activity and protein levels.

Long-Term Dynamic Changes of NMDA Receptors Following an Excitotoxic Challenge.

Excitotoxicity is a form of neuronal death characterized by the sustained activation of N-methyl-D-aspartate receptors (NMDARs) triggered by the excitatory neurotransmitter glutamate. NADPH-diaphorase neurons (also known as nNOS (+) neurons) are a subpopulation of aspiny interneurons, largely spared following excitotoxic challenges. Unlike nNOS (−) cells, nNOS (+) neurons fail to generate reactive oxygen species in response to NMDAR activation, a critical divergent step in the excitotoxic cascade. However, additional mechanisms underlying the reduced vulnerability of nNOS (+) neurons to NMDAR-driven neuronal death have not been explored. Using functional, genetic, and molecular analysis in striatal cultures, we indicate that nNOS (+) neurons possess distinct NMDAR properties. These specific features are primarily driven by the peculiar redox milieu of this subpopulation. In addition, we found that nNOS (+) neurons exposed to a pharmacological maneuver set to mimic chronic excitotoxicity alter their responses to NMDAR-mediated challenges. These findings suggest the presence of mechanisms providing long-term dynamic regulation of NMDARs that can have critical implications in neurotoxic settings.

Mislocalization of Nucleocytoplasmic Transport Proteins in Human Huntington's Disease PSC-Derived Striatal Neurons.

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene (HTT). Disease progression is characterized by the loss of vulnerable neuronal populations within the striatum. A consistent phenotype across HD models is disruption of nucleocytoplasmic transport and nuclear pore complex (NPC) function. Here we demonstrate that high content imaging is a suitable method for detecting mislocalization of lamin-B1, RAN and RANGAP1 in striatal neuronal cultures thus allowing a robust, unbiased, highly powered approach to assay nuclear pore deficits. Furthermore, nuclear pore deficits extended to the selectively vulnerable DARPP32 + subpopulation neurons, but not to astrocytes. Striatal neuron cultures are further affected by changes in gene and protein expression of RAN, RANGAP1 and lamin-B1. Lowering total HTT using HTT-targeted anti-sense oligonucleotides partially restored gene expression, as well as subtly reducing mislocalization of proteins involved in nucleocytoplasmic transport. This suggests that mislocalization of RAN, RANGAP1 and lamin-B1 cannot be normalized by simply reducing expression of CAG-expanded HTT in the absence of healthy HTT protein.

High-Content Single-Cell Förster Resonance Energy Transfer Imaging of Cultured Striatal Neurons Reveals Novel Cross-Talk in the Regulation of Nuclear Signaling by Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2.

Genetically encoded biosensors can be used to track signaling events in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. Here, we describe the use of genetically encoded biosensors based on Förster resonance energy transfer (FRET), combined with high-content microscopy, to image dynamic signaling events simultaneously in thousands of neurons in response to drug treatments. We first applied this approach to examine intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. Using high-content FRET imaging and immunofluorescence, we identified neuronal subpopulations with unique responses to pharmacological manipulation and used nuclear morphology to identify medium spiny neurons within these heterogeneous striatal cultures. Focusing on protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the cytoplasm and nucleus, we noted pronounced intercellular differences among putative medium spiny neurons, in both the magnitude and kinetics of signaling responses to drug application. Importantly, a conventional "bulk" analysis that pooled all cells in culture yielded a different rank order of drug potency than that revealed by single-cell analysis. Using a single-cell analytical approach, we dissected the relative contributions of PKA and ERK1/2 signaling in striatal neurons and unexpectedly identified a novel role for ERK1/2 in promoting nuclear activation of PKA in striatal neurons. This finding adds a new dimension of signaling crosstalk between PKA and ERK1/2 with relevance to dopamine D1 receptor signaling in striatal neurons. In conclusion, high-content single-cell imaging can complement and extend traditional population-level analyses and provides a novel vantage point from which to study cellular signaling. SIGNIFICANCE STATEMENT: High-content imaging revealed substantial intercellular variation in the magnitude and pattern of intracellular signaling events driven by receptor stimulation. Since individual neurons within the same population can respond differently to a given agonist, interpreting measures of intracellular signaling derived from the averaged response of entire neuronal populations may not always reflect what happened at the single-cell level. This study uses this approach to identify a new form of cross-talk between PKA and ERK1/2 signaling in the nucleus of striatal neurons.

The expression of tenascin-C in neural stem/progenitor cells is stimulated by the growth factors EGF and FGF-2, but not by TGF\u03b21

Neural stem/progenitor cells (NSPCs) rely on internal and external cues determining their lineage decisions during brain development. The progenitor cells of the embryonic mammalian forebrain reside in the ventricular and subventricular zones of the lateral ventricles, where they proliferate, generate neurons and glial cells, and respond to external cues like growth factors. The extracellular matrix (ECM) surrounds NSPCs and influences the cell fate by providing mechanical scaffold, trophic support, and instructive signals. The ECM molecule tenascin-C (Tnc) is expressed in the proliferative zones of the developing forebrain and involved in the proliferation and maturation of NSPCs. Here, we analyzed the regulation of the Tnc gene expression by NSPCs cultivated under the influence of different growth factors. We observed that the epidermal growth factor (EGF) and the fibroblast growth factor (FGF)-2 strongly increased the expression of Tnc, whereas the transforming growth factor (TGF)β 1 had no effect on Tnc gene expression, in contrast to previous findings in cell cultures of neural and non-neural origin. The stimulation of the Tnc gene expression induced by EGF or FGF-2 was reversible and seen in constantly treated as well as short term stimulated NSPC cultures. The activation depended on the presence of the respective receptors, which was slightly different in cortical and striatal NSPC cultures. Our results confirm the influence of extracellular stimuli regulating the expression of factors that form a niche for NSPCs during embryonic forebrain development.

Methamphetamine Blocks Adenosine A2A Receptor Activation via Sigma 1 and Cannabinoid CB1 Receptors.

Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A2A receptor (A2AR). First, we noticed that methamphetamine does not affect A2A functionality if the receptor is expressed in a heterologous system. However, A2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB1 receptor (CB1R) and the sigma 1 receptor (σ1R). Signaling via both adenosine A2AR and cannabinoid CB1R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A2AR–CB1R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A2AR- and the CB1R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σ1R, alters the expression and function of two interacting receptors, A2AR, which is a therapeutic target for neuroprotection, and CB1R, which is the most abundant G protein-coupled receptor (GPCR) in the brain.

Osteogenic Mechanisms of Basal Ganglia Calcification and its ex vivo Model in the Hypoparathyroid Milieu.

Basal-ganglia calcification (BGC) is common (70%) in patients with chronic hypoparathyroidism. Interestingly, cortical gray matter is spared from calcification. The mechanism of BGC, role of hyperphosphatemia, and modulation of osteogenic molecules by parathyroid hormone (PTH) in its pathogenesis is not clear. We assessed the expression of a large repertoire of molecules with proosteogenic or antiosteogenic effects, including neuroprogenitor cells in caudate, dentate, and cortical gray matter from normal autopsy tissues. The effect of high phosphate and PTH was assessed in an ex vivo model of BGC using striatum tissue culture of the Sprague-Dawley rat. The messenger RNA and protein expression of 39 molecules involved in multiple osteogenic pathways were assessed in 25 autopsy tissues using reverse-transcriptase polymerase chain reaction, Western blot, and immunofluorescence. The striatal culture was maintained in a hypoparathyroid milieu for 24 days with and without (a) high phosphate (10-mm β-glycerophosphate) and (b) PTH(1-34) (50 ng/mL Dulbecco's modified Eagle's medium-F12 media) for their effect on striatal calcification and osteogenic molecules. Procalcification molecules (osteonectin, β-catenin, klotho, FZD4, NT5E, LRP5, WNT3A, collagen-1α, and SOX2-positive neuroprogenitor stem cells) had significantly higher expression in the caudate than gray matter. Caudate nuclei also had higher expression of antiosteogenic molecules (osteopontin, carbonic anhydrase-II [CA-II], MGP, sclerostin, ISG15, ENPP1, and USP18). In an ex vivo model, striatum culture showed an increased propensity for calcified nodules with mineral deposition similar to that of bone tissue on Fourier-transformed infrared spectroscopy, alizarin, and von Kossa stain. Mineralization in striatal culture was enhanced by high phosphate and decreased by exogenous PTH through increased expression of CA-II. This study provides a conceptual advance on the molecular mechanisms of BGC and the possibility of PTH therapy to prevent this complication in a hypoparathyroid milieu.

Deficits in coordinated neuronal activity and network topology are striatal hallmarks in Huntington\u2019s disease

BackgroundNetwork alterations underlying neurodegenerative diseases often precede symptoms and functional deficits. Thus, their early identification is central for improved prognosis. In Huntington’s disease (HD), the cortico-striatal networks, involved in motor function processing, are the most compromised neural substrate. However, whether the network alterations are intrinsic of the striatum or the cortex is not fully understood.ResultsIn order to identify early HD neural deficits, we characterized neuronal ensemble calcium activity and network topology of HD striatal and cortical cultures. We used large-scale calcium imaging combined with activity-based network inference analysis. We extracted collective activity events and inferred the topology of the neuronal network in cortical and striatal primary cultures from wild-type and R6/1 mouse model of HD. Striatal, but not cortical, HD networks displayed lower activity and a lessened ability to integrate information. GABAA receptor blockade in healthy and HD striatal cultures generated similar coordinated ensemble activity and network topology, highlighting that the excitatory component of striatal system is spared in HD. Conversely, NMDA receptor activation increased individual neuronal activity while coordinated activity became highly variable and undefined. Interestingly, by boosting NMDA activity, we rectified striatal HD network alterations.ConclusionsOverall, our integrative approach highlights striatal defective network integration capacity as a major contributor of basal ganglia dysfunction in HD and suggests that increased excitatory drive may serve as a potential intervention. In addition, our work provides a valuable tool to evaluate in vitro network recovery after treatment intervention in basal ganglia disorders.

Dysregulated hydrogen sulfide metabolism links aberrant neuronal stress responses and neurodegeneration in Huntington's disease

Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays central roles in a myriad of physiological processes ranging from response to oxidative stress and transcriptional regulation. Hydrogen sulfide is synthesized endogenously from the semi‐essential amino acid cysteine via the reverse transsulfuration pathway. Thus disrupted cysteine metabolism may also lead to aberrant H2S signaling. One of the modes by which hydrogen sulfide signals is by a posttranslational modification designated as sulfhydration/persulfidation, which occurs on reactive cysteine residues on target proteins, where the reactive SH group is converted to an SSH group. Sulfhydration is a highly prevalent modification which modifies the structure and/or activity of target proteins. Research on H2S and persulfidation is still in its infancy. We show here that H2S is generated in response to stress stimuli such as Golgi stress and oxidative stress in neurons, which modulates sulfhydration levels to modulate signaling pathways. We further show that these cytoprotective stress responses are compromised in striatal cell culture model and the R6/2 and Q175 mouse models of Huntington’s disease (HD). We have shown previously that the biosynthetic enzyme for cysteine and H2S in neurons, cystathionine g‐lyase (CSE) is depleted in HD and mediates neurodegeneraton. HD is a neurodegenerative disease which is triggered by expansion of polyglutamine repeats in the protein huntingtin, which causes it to aggregate and elicit multi‐system toxicity. Restoring H2S balance in cells mitigates neuronal dysfunction in neurodegenerative diseases such as HD. A screen for regulators of the transsulfuration pathway has resulted in identification of compounds that modulate cysteine balance and disease progression in HD.Support or Funding InformationUnited States Public Health Service (USPHS) grants DA000266 and MH18501 to S.H.S.

Early epigenomic and transcriptional changes reveal Elk-1 transcription factor as a therapeutic target in Huntington’s disease

Huntington's disease (HD) is a chronic neurodegenerative disorder characterized by a late clinical onset despite ubiquitous expression of the mutant Huntingtin gene (HTT) from birth. Transcriptional dysregulation is a pivotal feature of HD. Yet, the genes that are altered in the prodromal period and their regulators, which present opportunities for therapeutic intervention, remain to be elucidated. Using transcriptional and chromatin profiling, we found aberrant transcription and changes in histone H3K27acetylation in the striatum of R6/1 mice during the presymptomatic disease stages. Integrating these data, we identified the Elk-1 transcription factor as a candidate regulator of prodromal changes in HD. Exogenous expression of Elk-1 exerted beneficial effects in a primary striatal cell culture model of HD, and adeno-associated virus-mediated Elk-1 overexpression alleviated transcriptional dysregulation in R6/1 mice. Collectively, our work demonstrates that aberrant gene expression precedes overt disease onset in HD, identifies the Elk-1 transcription factor as a key regulator linked to early epigenetic and transcriptional changes in HD, and presents evidence for Elk-1 as a target for alleviating molecular pathology in HD.

Characterization of Neurodevelopmental Abnormalities in iPSC-Derived Striatal Cultures from Patients with Huntington's Disease.

Background:Huntington’s disease (HD) is an inherited neurodegenerative disease and is characterized by atrophy of certain regions of the brain in a progressive manner. HD patients experience behavioral changes and uncontrolled movements which can be primarily attributed to the atrophy of striatal neurons. Previous publications describe the models of the HD striatum using induced pluripotent stem cells (iPSCs) derived from HD patients with a juvenile onset (JHD). In this model, the JHD iPSC-derived striatal cultures had altered neurodevelopment and contained a high number of nestin expressing progenitor cells at 42 days of differentiation.Objective:To further characterize the altered neurodevelopmental phenotype and evaluate potential phenotypic reversal.Methods:Differentiation of human iPSCs towards striatal fate and characterization by means of immunocytochemistry and stereological quantification.Results:Here this study demonstrates a distinct delay in the differentiation of the JHD neural progenitor population. However, reduction of the JHD aberrant progenitor populations can be accomplished either by targeting the canonical Notch signaling pathway or by treatment with HTT antisense oligonucleotides (ASOs).Conclusions:In summary, this data is postulated to reflect a potential overall developmental delay in JHD.

Endothelial Progenitor Cells Conditioned Medium Supports Number of GABAergic Neurons and Exerts Neuroprotection in Cultured Striatal Neuronal Progenitor Cells

There is growing evidence that stem and progenitor cells exert regenerative actions by means of paracrine factors. In line with these notions, we recently demonstrated that endothelial progenitor cell (EPC)-derived conditioned medium (EPC-CM) substantially increased viability of brain microvascular cells. In the present study, we aimed at investigating whether EPC-CM supports cell survival of cultured striatal progenitor cells. For that purpose, primary cultures from fetal rat embryonic (E14) ganglionic eminence were prepared and grown for 7 days in vitro (DIV). EPC-CM was administered from DIV5–7. Treatment of the striatal cultures with EPC-CM resulted in significantly increased densities of GABA-immunoreactive (-ir) neurons. Inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-kinase, but not of the ROCK pathway, significantly attenuated the EPC-CM induced increase in GABA-ir cell densities. Similar results were observed when EPC-CM was subjected to proteolytic digestion and lipid extraction. Furthermore, inhibition of translation abolished the EPC-CM induced effects. Importantly, EPC-CM displayed neuroprotection against 3-nitropropionic acid induced toxicity. These findings demonstrate that EPC-derived paracrine factors substantially promote survival and/or differentiation of cultured striatal progenitor cells involving both proteinaceous factors and lipidic factors. In sum, EPC-CM constituents might lead to a novel cell-free therapeutic strategy to challenge neuronal degeneration.

Brain-derived neurotrophic factor modulates neuronal network structure and dynamics in striatal cultures: Implications for Huntington's disease

Almost a third of the offspring of parents diagnosed with schizophrenia or bipolar disorder could develop a mental disorder or related symptoms. The objectives of this study were to test the existence of two distinct subgroups of youth at-risk, according to their retinal response to luminance measured with electroretinography (ERG), and to relate the resulting cluster memberships with the cognitive clusters previously reported.A clustering analysis was performed with ERG measurements in 107 at-risk offspring. Each subgroup was compared to a healthy control group of 203 individuals. The ERG subgroup memberships were then associated with the cognitive clusters.A two-cluster solution was obtained: HR-Cluster1 (n=53) showed a control-like ERG profile and HR-Cluster2 (n=54) showed reduced rod amplitudes and prolonged cone latencies of the b-wave. Subjects in the HR-Cluster2 were 2.7 times more likely to belong to the most detrimental cognitive subgroup than subjects in the HR-Cluster1 (49% Vs 18%).At-risk offspring showed two distinct ERG profiles: a control-like and an altered profile. A higher risk of impaired cognitive function was observed in subjects with the altered ERG profile, suggesting the ERG as a potential biomarker of susceptibility to mental illness among youth at risk.

Triglyceride Form of Docosahexaenoic Acid Mediates Neuroprotection in Experimental Parkinsonism.

Parkinson’s disease (PD) is a neurodegenerative disorder of unknown etiology. The main treatment of PD consists of medication with dopamine-based drugs, which palliate the symptoms but may produce adverse effects after chronic administration. Accordingly, there is a need to develop novel neuroprotective therapies. Several studies suggest that omega-3 polyunsaturated fatty acids (n-3 PUFA) might provide protection against brain damage. Here, we studied several experimental models of PD, using striatal neuronal cultures, striatal slices, and mice, to assess the neuroprotective effects of docosahexaenoic acid (DHA), the main n-3 PUFA in the brain, administered in its triglyceride form (TG-DHA). Hence, we determined the beneficial effects of TG-DHA on neural viability following 6-hydroxydopamine (6-OHDA)-induced neurotoxicity, a well-established PD model. We also implemented a novel mouse behavioral test, the beam walking test, to finely assess mouse motor skills following dopaminergic denervation. This test showed potential as a useful behavioral tool to assess novel PD treatments. Our results indicated that TG-DHA-mediated neuroprotection was independent of the net incorporation of PUFA into the striatum, thus suggesting a tight control of brain lipid homeostasis both in normal and pathological conditions.

Dnmt3a2 in the Nucleus Accumbens Shell Is Required for Reinstatement of Cocaine Seeking.

Epigenetic mechanisms have gained increasing attention as regulators of synaptic plasticity and responsiveness to drugs of abuse. In particular, it has been shown that the activity of the DNA methyltransferase 3a (Dnmt3a) mediates certain long-lasting effects of cocaine. Here we examined the role of the Dnmt isoforms, Dnmt3a1 and Dnmt3a2, within the nucleus accumbens (NAc) on transcriptional activity of immediate early genes (IEGs) and acute and long-lasting responsiveness to cocaine and cocaine conditioned cues. Using primary striatal cultures, we show that transcription of Dnmt3a2, but not that of Dnmt3a1, is activated by dopamine D1 receptor signaling and that knockdown of Dnmt3a2 using viral vector-mediated expression of Dnmt3a2-specific shRNAs impairs induction of the IEGs, Arc, FosB, and Egr2 Acute cocaine administration increases expression of Dnmt3a2 but not that of Dnmt3a1 in the NAc shell. In contrast, in the NAc core, expression of Dnmt3a1 and Dnmt3a2 was unaffected by cocaine administration. shRNA-mediated knockdown of Dnmt3a2 in vivo impairs the induction of IEGs, including Egr2 and FosB indicating that Dnmt3a2 regulates cocaine-dependent expression of plasticity genes in the rat NAc shell. Cocaine self-administration experiments in rats revealed that Dnmt3a2 regulates drug cue memories that drive reinstatement of cocaine seeking as well as incubation of this phenomenon within the NAc shell. Dnmt3a2 does not influence the primary reinforcing effects of cocaine. Thus, Dnmt3a2 mediates long-lasting cocaine cue memories within the NAc shell. Targeting Dnmt3a2 expression or function may interfere with cocaine craving and relapse.SIGNIFICANCE STATEMENT In humans, drug craving can occur in response to conditioned cues, even after extended periods of abstinence. In rats, cue-induced cocaine seeking has been shown to increase progressively during the first 2 months of abstinence from drug self-administration. This phenomenon, referred to as incubation of cocaine seeking, is consistent with the hypothesis that in humans craving increases over time and remains high following prolonged abstinence. Those long-lasting behavioral changes are likely to be mediated by epigenetic effects and neuroplastic changes within the mesolimbic brain reward system. Here we show that a specific isoform of DNA-methyltransferases in the NAc shell regulates drug cue memories that drive reinstatement of cocaine seeking after both early abstinence and incubation of cocaine craving.

Striatin-1 is a B subunit of protein phosphatase PP2A that regulates dendritic arborization and spine development in striatal neurons

Striatin-1, a subunit of the serine/threonine phosphatase PP2A, is preferentially expressed in neurons in the striatum. As a member of the striatin family of B subunits, striatin-1 is a core component together with PP2A of a multiprotein complex called STRIPAK, the striatin-interacting phosphatase and kinase complex. Little is known about the function of striatin-1 or the STRIPAK complex in the mammalian striatum. Here, we identify a selective role for striatin-1 in striatal neuron maturation. Using a small hairpin RNA (shRNA) knockdown approach in primary striatal neuronal cultures, we determined that reduced expression of striatin-1 results in increased dendritic complexity and an increased density of dendritic spines, classified as stubby spines. The dendritic phenotype was rescued by co-expression of a striatin-1 mutant construct insensitive to the knockdown shRNA but was not rescued by co-expression of PP2A- or Mob3-binding deficient striatin-1 constructs. Reduction of striatin-1 did not result in deficits in neuronal connectivity in this knockdown model, as we observed no abnormalities in synapse formation or in spontaneous excitatory postsynaptic currents. Thus, this study suggests that striatin-1 is a regulator of neuronal development in striatal neurons.