Dopamine D2R is Required for Hippocampal-dependent Memory and Plasticity at the CA3-CA1 Synapse.

Dopamine receptors play an important role in motivational, emotional, and motor responses. In addition, growing evidence suggests a key role of hippocampal dopamine receptors in learning and memory. It is well known that associative learning and synaptic plasticity of CA3-CA1 requires the dopamine D1 receptor (D1R). However, the specific role of the dopamine D2 receptor (D2R) on memory-related neuroplasticity processes is still undefined. Here, by using two models of D2R loss, D2R knockout mice (Drd2-/-) and mice with intrahippocampal injections of Drd2-small interfering RNA (Drd2-siRNA), we aimed to investigate how D2R is involved in learning and memory as well as in long-term potentiation of the hippocampus. Our studies revealed that the genetic inactivation of D2R impaired the spatial memory, associative learning, and the classical conditioning of eyelid responses. Similarly, deletion of D2R reduced the activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. Our results demonstrate the first direct evidence that D2R is essential in behaving mice for trace eye blink conditioning and associated changes in hippocampal synaptic strength. Taken together, these results indicate a key role of D2R in regulating hippocampal plasticity changes and, in consequence, acquisition and consolidation of spatial and associative forms of memory.

Beneficial effects of the phytocannabinoid Δ9-THCV in L-DOPA-induced dyskinesia in Parkinson's disease.

The antioxidant and CB2 receptor agonist properties of Δ9-tetrahydrocannabivarin (Δ9-THCV) afforded neuroprotection in experimental Parkinson's disease (PD), whereas its CB1 receptor antagonist profile at doses lower than 5 mg/kg caused anti-hypokinetic effects. In the present study, we investigated the anti-dyskinetic potential of Δ9-THCV (administered i.p. at 2 mg/kg for two weeks), which had not been investigated before. This objective was investigated after inducing dyskinesia by repeated administration of L-DOPA (i.p. at 10 mg/kg) in a genetic model of dopaminergic deficiency, Pitx3ak mutant mice, which serves as a useful model for testing anti-dyskinetic agents. The daily treatment of these mice with L-DOPA for two weeks progressively increased the time spent in abnormal involuntary movements (AIMs) and elevated their horizontal and vertical activities (as measured in a computer-aided actimeter), signs that reflected the dyskinetic state of these mice. Interestingly, when combined with L-DOPA from the first injection, Δ9-THCV delayed the appearance of all these signs and decreased their intensity, with a reduction in the levels of FosB protein and the histone pAcH3 (measured by immunohistochemistry), which had previously been found to be elevated in the basal ganglia in L-DOPA-induced dyskinesia. In addition to the anti-dyskinetic effects of Δ9-THCV when administered at the onset of L-DOPA treatment, Δ9-THCV was also effective in attenuating the intensity of dyskinesia when administered for three consecutive days once these signs were already present (two weeks after the onset of L-DOPA treatment). In summary, our data support the anti-dyskinetic potential of Δ9-THCV, both to delay the occurrence and to attenuate the magnitude of dyskinetic signs. Although further studies are clearly required to determine the clinical significance of these data in humans, the results nevertheless situate Δ9-THCV in a promising position for developing a cannabinoid-based therapy for patients with PD.

Emerging roles for long noncoding RNAs in learning, memory and associated disorders.

While protein-coding genes have been widely studied in learning and memory, the role of the non-coding genome has only recently been investigated. With advances in high throughput sequencing technologies and functional profiling methods, multiple long noncoding RNAs (lncRNAs) have been functionally and mechanistically linked with neurobiological processes related with learning and memory, as well disorders that lead to memory impairment. However, these macromolecules are still a subject of controversy and intense scrutiny regarding the proper criteria for determining their functionality and their evolution in the central nervous system. Recent studies have implicated multiple lncRNAs as critical regulators of gene expression in the central nervous system and mediate learning processes. In this review, we explore possible explanations for how lncRNAs are evolved in our central nervous system, discuss our current understanding of their involvement in learning and memory related disorders, and describe emerging tools for studying lncRNAs.

N370S-GBA1 mutation causes lysosomal cholesterol accumulation in Parkinson's disease.

BACKGROUND: Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme ß-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. METHODS: We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. RESULTS: The N370S mutation produced a significant reduction in ß-glucocerebrosidase-1 protein and enzyme activity and ß-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. CONCLUSIONS: Our results support a connection between the loss of ß-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society.

Nitric oxide synthase inhibition decreases l-DOPA-induced dyskinesia and the expression of striatal molecular markers in Pitx3(-/-) aphakia mice.

Nitric oxide (NO), a gaseous messenger molecule synthesized by nitric oxide synthase (NOS), plays a pivotal role in integrating dopamine transmission in the basal ganglia and has been implicated in the pathogenesis of Parkinson disease (PD). To study the role of the nitrergic system in l-DOPA-induced dyskinesia (LID), we assessed the effect of the pharmacological manipulation of NO levels and NO/cyclic guanosine monophosphate (cGMP) signaling on LID in the Pitx3(-/-) aphakia mouse, a genetic model of PD. To evaluate the effect of decreased NO signaling on the development of LID, Pitx3(-/-) mice were chronically treated with l-DOPA and 7-nitroindazole (7-NI, a neuronal NOS inhibitor). To evaluate its effect on the expression of established LID, 7-NI was administered acutely to dyskinetic mice. The chronic 7-NI treatment attenuated the development of LID in the Pitx3(-/-) mice, and the sub-acute 7-NI treatment attenuated established dyskinesia without affecting the beneficial therapeutic effect of l-DOPA. Moreover, 7-NI significantly reduced FosB and pAcH3 expression in the acutely and chronically l-DOPA-treated mice. We also examined how increasing NO/cGMP signaling affects LID expression by acutely administering molsidomine (an NO donor) or zaprinast (a cGMP phosphodiesterase 5-PDE5 inhibitor) before l-DOPA in mice with established dyskinesia. Paradoxically, the administration of either of these drugs also significantly diminished the expression of established LID; however, the effect occurred at the expense of the antiparkinsonian l-DOPA properties. We demonstrate that targeting the NO/cGMP signaling pathway reduces dyskinetic behaviors and molecular markers, but only the 7-NI treatment preserved the antiparkinsonian effect of l-DOPA, indicating that NOS inhibitors represent a potential therapy to reduce LID.

Synaptically-targeted long non-coding RNA SLAMR promotes structural plasticity by increasing translation and CaMKII activity.

Long noncoding RNAs (lncRNAs) play crucial roles in maintaining cell homeostasis and function. However, it remains largely unknown whether and how neuronal activity impacts the transcriptional regulation of lncRNAs, or if this leads to synapse-related changes and contributes to the formation of long-term memories. Here, we report the identification of a lncRNA, SLAMR, which becomes enriched in CA1-hippocampal neurons upon contextual fear conditioning but not in CA3 neurons. SLAMR is transported along dendrites via the molecular motor KIF5C and is recruited to the synapse upon stimulation. Loss of function of SLAMR reduces dendritic complexity and impairs activity-dependent changes in spine structural plasticity and translation. Gain of function of SLAMR, in contrast, enhances dendritic complexity, spine density, and translation. Analyses of the SLAMR interactome reveal its association with CaMKIIα protein through a 220-nucleotide element also involved in SLAMR transport. A CaMKII reporter reveals a basal reduction in CaMKII activity with SLAMR loss-of-function. Furthermore, the selective loss of SLAMR function in CA1 disrupts the consolidation of fear memory in male mice, without affecting their acquisition, recall, or extinction, or spatial memory. Together, these results provide new molecular and functional insight into activity-dependent changes at the synapse and consolidation of contextual fear.

Hydroxycitrate delays early mortality in mice and promotes muscle regeneration while inducing a rich hepatic energetic status.

ATP citrate lyase (ACLY) inhibitors have the potential of modulating central processes in protein, carbohydrate, and lipid metabolism, which can have relevant physiological consequences in aging and age-related diseases. Here, we show that hepatic phospho-active ACLY correlates with overweight and Model for End-stage Liver Disease score in humans. Wild-type mice treated chronically with the ACLY inhibitor potassium hydroxycitrate exhibited delayed early mortality. In AML12 hepatocyte cultures, the ACLY inhibitors potassium hydroxycitrate, SB-204990, and bempedoic acid fostered lipid accumulation, which was also observed in the liver of healthy-fed mice treated with potassium hydroxycitrate. Analysis of soleus tissue indicated that potassium hydroxycitrate produced the modulation of wound healing processes. In vivo, potassium hydroxycitrate modulated locomotor function toward increased wire hang performance and reduced rotarod performance in healthy-fed mice, and improved locomotion in mice exposed to cardiotoxin-induced muscle atrophy. Our findings implicate ACLY and ACLY inhibitors in different aspects of aging and muscle regeneration.

Involvement of cannabinoid CB1 receptor in associative learning and in hippocampal CA3-CA1 synaptic plasticity.

We studied, in behaving mice, the contribution of CB1 receptors to the activity-dependent changes induced at the hippocampal CA3-CA1 synapse by associative learning and following experimentally evoked long-term potentiation (LTP). Mice were classically conditioned to evoke eyelid responses with a trace paradigm using a tone as conditioned stimulus (CS) and an electric shock as unconditioned stimulus (US). Field excitatory postsynaptic potentials (fEPSPs) were evoked at the CA3-CA1 synapse during the CS-US interval across training. Conditioning was performed in presence of an agonist (WIN55,212-2) alone or with an antagonist (AM251) of the CB1 receptor, injected either systemically or locally. Conditioned responses (CRs) and fEPSP potentiation were depressed by WIN55,212-2. LTP was evoked by high-frequency stimulation of Schaffer collaterals after systemic or local WIN55,212-2 and AM251 injections. WIN55,212-2 affected the induction phase of LTP, mainly when injected locally. The addition of AM251 canceled out the effects of WIN55,212-2. Similar experiments were carried out in animals lacking the CB1 receptor (CB1(-/-) mice) and following silencing of hippocampal CB1 receptors (CB1R-siRNA-injected animals). In this case, CRs (CB1(-/-) mice) and LTP (CB1(-/-) and CB1R-siRNA-injected mice) reached lower values than their respective controls. Results offer new insights for understanding CB1 receptor contribution to associative learning and to CA3-CA1 synaptic plasticity.

SLAMR, a synaptically targeted lncRNA, facilitates the consolidation of contextual fear memory.

LncRNAs are involved in critical processes for cell homeostasis and function. However, it remains largely unknown whether and how the transcriptional regulation of long noncoding RNAs results in activity-dependent changes at the synapse and facilitate formation of long-term memories. Here, we report the identification of a novel lncRNA, SLAMR, that becomes enriched in CA1- but not in CA3-hippocampal neurons upon contextual fear conditioning. SLAMR is transported to dendrites via the molecular motor KIF5C and recruited to the synapse in response to stimulation. Loss of function of SLAMR reduced dendritic complexity and impaired activity dependent changes in spine structural plasticity. Interestingly, gain of function of SLAMR enhanced dendritic complexity, and spine density through enhanced translation. Analyses of the SLAMR interactome revealed its association with CaMKIIα protein through a 220-nucleotide element and its modulation of CaMKIIα activity. Furthermore, loss-of-function of SLAMR in CA1 selectively impairs consolidation but neither acquisition, recall, nor extinction of fear memory and spatial memory. Together, these results establish a new mechanism for activity dependent changes at the synapse and consolidation of contextual fear.

Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.

Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.

Metabolic reprogramming by Acly inhibition using SB-204990 alters glucoregulation and modulates molecular mechanisms associated with aging.

ATP-citrate lyase is a central integrator of cellular metabolism in the interface of protein, carbohydrate, and lipid metabolism. The physiological consequences as well as the molecular mechanisms orchestrating the response to long-term pharmacologically induced Acly inhibition are unknown. We report here that the Acly inhibitor SB-204990 improves metabolic health and physical strength in wild-type mice when fed with a high-fat diet, while in mice fed with healthy diet results in metabolic imbalance and moderated insulin resistance. By applying a multiomic approach using untargeted metabolomics, transcriptomics, and proteomics, we determined that, in vivo, SB-204990 plays a role in the regulation of molecular mechanisms associated with aging, such as energy metabolism, mitochondrial function, mTOR signaling, and folate cycle, while global alterations on histone acetylation are absent. Our findings indicate a mechanism for regulating molecular pathways of aging that prevents the development of metabolic abnormalities associated with unhealthy dieting. This strategy might be explored for devising therapeutic approaches to prevent metabolic diseases.

L-DOPA-induced increase in TH-immunoreactive striatal neurons in parkinsonian mice: insights into regulation and function.

Tyrosine hydroxylase (TH)-immunoreactive (ir) neurons have been found in the striatum after dopamine depletion; however, little is known about the mechanism underlying their appearance or their functional significance. We previously showed an increase in striatal TH-ir neurons after L-DOPA treatment in mice with unilateral 6-OHDA lesions in the striatum. In the present study, we further examined the time-course and persistence of the effects of chronic L-DOPA treatment on the appearance and regulation of TH-ir neurons as well as their possible function. We found that the L-DOPA-induced increase in striatal TH-ir neurons is dose-dependent and persists for days after L-DOPA withdrawal, decreasing significantly 10 days after L-DOPA treatment ends. Using hemiparkinsonian D1 receptor knock-out (D1R-/-) and D2 receptor knock-out (D2R-/-) mice, we found that the D1R, but not the D2R, is required for the L-DOPA-induced appearance of TH-ir neurons in the dopamine-depleted striatum. Interestingly, our experiments in aphakia mice, which lack Pitx3 expression in the brain, indicate that the L-DOPA-dependent increase in the number of TH-ir neurons is independent of Pitx3, a transcription factor necessary for the development of mesencephalic dopaminergic neurons. To explore the possible function of L-DOPA-induced TH-ir neurons in the striatum, we examined dopamine overflow and forelimb use in L-DOPA-treated parkinsonian mice. These studies revealed a tight spatio-temporal correlation between the presence of striatal TH-ir neurons, the recovery of electrically stimulated dopamine overflow in the lesioned striatum, and the recovery of contralateral forelimb use with chronic L-DOPA treatment. Our results suggest that the presence of TH-ir neurons in the striatum may underlie the long-duration response to L-DOPA following withdrawal. Promotion of these neurons in the early stages of Parkinson's disease, when dopamine denervation is incomplete, may be beneficial for maintaining motor function.

Associative learning and CA3-CA1 synaptic plasticity are impaired in D1R null, Drd1a-/- mice and in hippocampal siRNA silenced Drd1a mice.

Associative learning depends on multiple cortical and subcortical structures, including striatum, hippocampus, and amygdala. Both glutamatergic and dopaminergic neurotransmitter systems have been implicated in learning and memory consolidation. While the role of glutamate is well established, the role of dopamine and its receptors in these processes is less clear. In this study, we used two models of dopamine D(1) receptor (D(1)R, Drd1a) loss, D(1)R knock-out mice (Drd1a(-/-)) and mice with intrahippocampal injections of Drd1a-siRNA (small interfering RNA), to study the role of D(1)R in different models of learning, hippocampal long-term potentiation (LTP) and associated gene expression. D(1)R loss markedly reduced spatial learning, fear learning, and classical conditioning of the eyelid response, as well as the associated activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. These results provide the first experimental demonstration that D(1)R is required for trace eyeblink conditioning and associated changes in synaptic strength in hippocampus of behaving mice. Drd1a-siRNA mice were indistinguishable from Drd1a(-/-) mice in all experiments, indicating that hippocampal knockdown was as effective as global inactivation and that the observed effects are caused by loss of D(1)R and not by indirect developmental effects of Drd1a(-/-). Finally, in vivo LTP and LTP-induced expression of Egr1 in the hippocampus were significantly reduced in Drd1a(-/-) and Drd1a-siRNA, indicating an important role for D(1)R in these processes. Our data reveal a functional relationship between acquisition of associative learning, increase in synaptic strength at the CA3-CA1 synapse, and Egr1 induction in the hippocampus by demonstrating that all three are dramatically impaired when D(1)R is eliminated or reduced.

Molecular motor KIF3B in the prelimbic cortex constrains the consolidation of contextual fear memory.

Molecular and cellular mechanisms underlying the role of the prelimbic cortex in contextual fear memory remain elusive. Here we examined the kinesin family of molecular motor proteins (KIFs) in the prelimbic cortex for their role in mediating contextual fear, a form of associative memory. KIFs function as critical mediators of synaptic transmission and plasticity by their ability to modulate microtubule function and transport of gene products. However, the regulation and function of KIFs in the prelimbic cortex insofar as mediating memory consolidation is not known. We find that within one hour of contextual fear conditioning, the expression of KIF3B is upregulated in the prelimbic but not the infralimbic cortex. Importantly, lentiviral-mediated knockdown of KIF3B in the prelimbic cortex produces deficits in consolidation while reducing freezing behavior during extinction of contextual fear. We also find that the depletion of KIF3B increases spine density within prelimbic neurons. Taken together, these results illuminate a key role for KIF3B in the prelimbic cortex as far as mediating contextual fear memory.

Activity-regulated synaptic targeting of lncRNA ADEPTR mediates structural plasticity by localizing Sptn1 and AnkB in dendrites.

Activity-dependent structural plasticity at the synapse requires specific changes in the neuronal transcriptome. While much is known about the role of coding elements in this process, the role of the long noncoding transcriptome remains elusive. Here, we report the discovery of an intronic long noncoding RNA (lncRNA)-termed ADEPTR-that is up-regulated and synaptically transported in a cAMP/PKA-dependent manner in hippocampal neurons, independently of its protein-coding host gene. Loss of ADEPTR function suppresses activity-dependent changes in synaptic transmission and structural plasticity of dendritic spines. Mechanistically, dendritic localization of ADEPTR is mediated by molecular motor protein Kif2A. ADEPTR physically binds to actin-scaffolding regulators ankyrin (AnkB) and spectrin (Sptn1) via a conserved sequence and is required for their dendritic localization. Together, this study demonstrates how activity-dependent synaptic targeting of an lncRNA mediates structural plasticity at the synapse.

Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation.

Synaptic structural plasticity, key to long-term memory storage, requires translation of localized RNAs delivered by long-distance transport from the neuronal cell body. Mechanisms and regulation of this system remain elusive. Here, we explore the roles of KIF5C and KIF3A, two members of kinesin superfamily of molecular motors (Kifs), and find that loss of function of either kinesin decreases dendritic arborization and spine density whereas gain of function of KIF5C enhances it. KIF5C function is a rate-determining component of local translation and is associated with ∼650 RNAs, including EIF3G, a regulator of translation initiation, and plasticity-associated RNAs. Loss of function of KIF5C in dorsal hippocampal CA1 neurons constrains both spatial and contextual fear memory, whereas gain of function specifically enhances spatial memory and extinction of contextual fear. KIF5C-mediated long-distance transport of local translation substrates proves a key mechanism underlying structural plasticity and memory.

Methamphetamine causes degeneration of dopamine cell bodies and terminals of the nigrostriatal pathway evidenced by silver staining.

Methamphetamine is a widely abused illicit drug. Recent epidemiological studies showed that methamphetamine increases the risk for developing Parkinson's disease (PD) in agreement with animal studies showing dopaminergic neurotoxicity. We examined the effect of repeated low and medium doses vs single high dose of methamphetamine on degeneration of dopaminergic terminals and cell bodies. Mice were given methamphetamine in one of the following paradigms: three injections of 5 or 10 mg/kg at 3 h intervals or a single 30 mg/kg injection. The integrity of dopaminergic fibers and cell bodies was assessed at different time points after methamphetamine by tyrosine hydroxylase immunohistochemistry and silver staining. The 3 × 10 protocol yielded the highest loss of striatal dopaminergic terminals, followed by the 3 × 5 and 1 × 30. Some degenerating axons could be followed from the striatum to the substantia nigra pars compacta (SNpc). All protocols induced similar significant degeneration of dopaminergic neurons in the SNpc, evidenced by amino-cupric-silver-stained dopaminergic neurons. These neurons died by necrosis and apoptosis. Methamphetamine also killed striatal neurons. By using D1-Tmt/D2-GFP BAC transgenic mice, we observed that degenerating striatal neurons were equally distributed between direct and indirect medium spiny neurons. Despite the reduced number of dopaminergic neurons in the SNpc at 30 days after treatment, there was a partial time-dependent recovery of dopamine terminals beginning 3 days after treatment. Locomotor activity and motor coordination were robustly decreased 1-3 days after treatment, but recovered at later times along with dopaminergic terminals. These data provide direct evidence that methamphetamine causes long-lasting loss/degeneration of dopaminergic cell bodies in the SNpc, along with destruction of dopaminergic terminals in the striatum.