Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage λ domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the λ domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2-E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera-accelerated internalization, impaired recycling, and endosomal sorting complex required for transport-dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases.

The process of locomotion is controlled by fine-tuned dopaminergic neurons in the Substantia Nigra pars-compacta (SNpc) that projects their axons to the dorsal striatum regulating cortical innervations of medium spiny neurons. Dysfunction in dopaminergic neurotransmission within the striatum leads to movement impairments, gaiting defects, and hypo-locomotion. Due to their high polarity and extreme axonal arborization, neurons depend on molecular motor proteins and microtubule-based transport for their normal function. Transport defects have been associated with neurodegeneration since axonopathies, axonal clogging, microtubule destabilization, and lower motor proteins levels were described in the brain of patients with Parkinson's Disease and other neurodegenerative disorders. However, the contribution of specific motor proteins to the regulation of the nigrostriatal network remains unclear. Here, we generated different conditional knockout mice for the kinesin heavy chain 5B subunit (Kif5b) of Kinesin-1 to unravel its contribution to locomotion. Interestingly, mice with neuronal Kif5b deletion showed hypo-locomotion, movement initiation deficits, and coordination impairments. High pressure liquid chromatography determined that dopamine (DA) metabolism is impaired in neuronal Kif5b-KO, while no dopaminergic cell loss was observed. However, the deletion of Kif5b only in dopaminergic neurons is not sufficient to induce locomotor defects. Noteworthy, pharmacological stimulation of DA release together with agonist or antagonist of DA receptors revealed selective D2-dependent movement initiation defects in neuronal Kif5b-KO. Finally, subcellular fractionation from striatum showed that Kif5b deletion reduced the amount of dopamine D2 receptor in synaptic plasma membranes. Together, these results revealed an important role for Kif5b in the modulation of the striatal network that is relevant to the overall locomotor response. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

The dopamine transporter (DAT) is a plasma membrane transport protein responsible for regulating the duration and intensity of dopaminergic signaling. Altered expression of DAT is linked to neurodevelopmental disorders, including attention deficit hyperactivity disorder and autism spectrum disorder, and is shown to contribute to the response of psychotropic drugs and neurotoxicants. Although the postnatal levels of DAT have been characterized, there are few data regarding the mechanisms that regulate postnatal DAT expression. Here, we examine the ontogeny of DAT mRNA from postnatal days 0 to 182 in the rat brain and define a role for epigenetic mechanisms regulating DAT expression. DAT mRNA and protein significantly increased between PND 0 and 6 months in rat midbrain and striatum, respectively. The epigenetic modifiers Dnmt1, Dnmt3a, Dnmt3b, and Hdac2 demonstrated age associated decreases in mRNA expression whereas Hdac5 and Hdac8 showed increased mRNA expression with age. Chromatin immunoprecipitation studies revealed increased protein enrichment of acetylated histone 3 at lysines 9 and 14 and the dopaminergic transcription factors Nurr1 and Pitx3 within the DAT promoter in an age-related manner. Together these studies provide evidence for the role of epigenetic modifications in the regulation of DAT during development. The identification of these mechanisms may contribute to potential therapeutic interventions aimed at neurodevelopmental disorders of the dopaminergic system.

Monoamine transporters (MATs) carry out neurotransmitter reuptake from the synaptic cleft, a key step in neurotransmission, which is targeted in the treatment of neurological disorders. Cholesterol (CHOL), a major component of the synaptic plasma membrane, has been shown to exhibit a modulatory effect on MATs. Recent crystal structures of the dopamine transporter (DAT) revealed the presence of two conserved CHOL-like molecules, suggesting a functional protein-CHOL direct interaction. Here, we present extensive atomistic molecular dynamics (MD) simulations of DAT in an outward-facing conformation. In the absence of bound CHOL, DAT undergoes structural changes reflecting early events of dopamine transport: transition to an inward-facing conformation. In contrast, in the presence of bound CHOL, these conformational changes are inhibited, seemingly by an immobilization of the intracellular interface of transmembrane helix 1a and 5 by CHOL. We also provide evidence, from coarse grain MD simulations that the CHOL sites observed in the DAT crystal structures are preserved in all human monoamine transporters (dopamine, serotonin and norepinephrine), suggesting that our findings might extend to the entire family.

Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNS-the Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions.

Fragile X Syndrome, a leading cause of inherited intellectual disability and autism, arises from transcriptional silencing of the FMR1 gene encoding an RNA-binding protein, Fragile X Mental Retardation Protein (FMRP). FMRP can regulate the expression of approximately 4% of brain transcripts through its role in regulation of mRNA transport, stability and translation, thus providing a molecular rationale for its potential pleiotropic effects on neuronal and brain circuitry function. Several intracellular signaling pathways are dysregulated in the absence of FMRP suggesting that cellular deficits may be broad and could result in homeostatic changes. Lipid rafts are specialized regions of the plasma membrane, enriched in cholesterol and glycosphingolipids, involved in regulation of intracellular signaling. Among transcripts targeted by FMRP, a subset encodes proteins involved in lipid biosynthesis and homeostasis, dysregulation of which could affect the integrity and function of lipid rafts. Using a quantitative mass spectrometry-based approach we analyzed the lipid raft proteome of Fmr1 knockout mice, an animal model of Fragile X syndrome, and identified candidate proteins that are differentially represented in Fmr1 knockout mice lipid rafts. Furthermore, network analysis of these candidate proteins reveals connectivity between them and predicts functional connectivity with genes encoding components of myelin sheath, axonal processes and growth cones. Our findings provide insight to aid identification of molecular and cellular dysfunctions arising from Fmr1 silencing and for uncovering shared pathologies between Fragile X syndrome and other autism spectrum disorders.

Astrocytes contain transport systems that are capable of removing various neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Glial serotonin transporter (SERT) plays an important role in the re-uptake of 5-hydroxytryptamine (5-HT). We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins using specific site-directed monoclonal antibodies (MoAb). Furthermore, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method, we addressed the expression of SERT mRNA in cultured rat astrocytes. We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [3H]5-HT into cultured astrocytes and cortical synaptosomes. Tricyclic antidepressants (clomipramine and imipramine) as well as selective serotonin re-uptake inhibitors (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [3H]5-HT uptake in both preparations. In contrast, the inhibitory effects of NE uptake inhibitors (nisoxetine and desipramine) and cocaine were weaker than those of 5-HT uptake inhibitors. In addition, dopamine (DA) uptake inhibitors (nomifensine and GBR-12935) exhibited a Ki value in the low micromolar range. The inhibitory potencies were in the order 5-HT uptake inhibitors (clomipramine, fluvoxamine, fluoxetine, imipramine and zimelidine) > NE uptake inhibitors (nisoxetine and desipramine) = cocaine > DA uptake inhibitors (nomifensine and GBR-12935). There was no difference in the order of the inhibitory effects of various monoamine uptake inhibitors between the two preparations. A correlation analysis of the potencies of various monoamine uptake inhibitors in the inhibition of [3H]5-HT into cultured astrocytes and cortical synaptosomes produced a highly significant correlation coefficient of 0.9893 (P < 0.0001). Immunocytochemical staining using anti-SERT MoAb in cultured astrocytes revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or golgi membranes, showed a considerable level of immunoreactivity. Extracts of astrocytes and synaptosomes from the cortex were immunoblotted with anti-SERT MoAb. SDS-PAGE/Western blots indicate that anti-SERT MoAb recognized two bands of 120 and 73 kDa in both preparations. RT-PCR demonstrated that astrocytes in cultured expressed mRNA for the cloned SERT protein, which has been characterized as the neuronal SERT. These pharmacological experiments indicate that this uptake process takes place through glial SERT that is very similar to neuronal SERT. Furthermore, the present data also indicate that the presence of the mRNA and protein for the neuronal SERT were established in cultured rat astrocytes, and the polypeptide portion of SERT in astrocytes and frontal cortex could be the same gene product.

Changes in serotonin transporter (SERT) function have been implicated in autism. SERT function is influenced by the number of transporter molecules present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. Thus, we searched for novel SERT-binding proteins and investigated whether the expression of one such protein was affected in subjects with autism.

The importance of in silico predictions for understanding cellular processes is now widely accepted, and a variety of algorithms useful for studying different biological features have been designed. In particular, the prediction of cis regulatory modules in non-coding human genome regions represents a major challenge for understanding gene regulation in several diseases. Recently, studies of the Wnt signaling pathway revealed a connection with neurodegenerative diseases such as Alzheimer's. In this article, we construct a classification tool that uses the transcription factor binding site motifs composition of some gene promoters to identify new Wnt/beta-catenin pathway target genes potentially involved in brain diseases.