Clozapine Induces Astrocyte-Dependent FDG-PET Hypometabolism

Purpose Advances in functional imaging allowed us to visualize brain glucose metabolism in vivo and non-invasively with [18F]fluoro-2-deoxyglucose (FDG) positron emission tomography (PET) imaging. In the past decades, FDG-PET has been instrumental in the understanding of brain function in health and disease. The source of the FDG-PET signal has been attributed to neuronal uptake, with hypometabolism being considered as a direct index of neuronal dysfunction or death. However, other brain cells are also metabolically active, including astrocytes. Based on the astrocyte-neuron lactate shuttle hypothesis, the activation of the glutamate transporter 1 (GLT-1) acts as a trigger for glucose uptake by astrocytes. With this in mind, we investigated glucose utilization changes after pharmacologically downregulating GLT-1 with clozapine (CLO), an antipsychotic drug. Methods Adult male Wistar rats (control, n = 14; CLO, n = 12) received CLO (25/35mg kg−1) for six weeks. CLO effects were evaluated in vivo with FDG-PET and cortical tissue was used to evaluate glutamate uptake, GLT-1 and GLAST levels. CLO treatment effects were also assessed in cortical astrocyte cultures (glucose and glutamate uptake, GLT-1 and GLAST levels) and in cortical neuronal cultures (glucose uptake). Results CLO markedly reduced in vivo brain glucose metabolism in several brain areas, especially in the cortex. Ex vivo analyses demonstrated decreased cortical glutamate transport along with GLT-1 mRNA and protein downregulation. In astrocyte cultures, CLO decreased GLT-1 density as well as glutamate and glucose uptake. By contrast, in cortical neuronal cultures, CLO did not affect glucose uptake. Conclusion This work provides in vivo demonstration that GLT-1 downregulation induces astrocyte-dependent cortical FDG-PET hypometabolism - mimicking the hypometabolic signature seen in people developing dementia - and adds further evidence that astrocytes are key contributors of the FDG-PET signal.

New Serotonin-Norepinephrine Reuptake Inhibitors and Their Anesthetic and Analgesic Considerations

Serotonin-norepinephrine reuptake inhibitors (SNRIs) inhibit the presynaptic neuronal uptake of serotonin and norepinephrine and prolong the effects of the monoamines in the synaptic cleft within the central nervous system, leading to increased postsynaptic receptor activation and neuronal activities. Serotonin-norepinephrine reuptake inhibitors can have multiple clinical indications, including as the first-line agents for the management of depression and anxiety, and as analgesics in the treatment of chronic pain. The effects of reuptake inhibition of norepinephrine and serotonin are often dose-dependent and agent-dependent. There are five FDA-approved serotonin-norepinephrine reuptake inhibitors (desvenlafaxine, duloxetine, levomilnacipran, milnacipran and sibutramine) currently being marketed in the United States. As the COVID-19 pandemic significantly increased the incidence and prevalence of anxiety and depression across the country, there are significantly increased prescriptions of these medications perioperatively. Thus, anesthesiologists are more likely than ever to have patients administered with these agents and scheduled for elective or emergency surgical procedures. A thorough understanding of these commonly prescribed serotonin-norepinephrine reuptake inhibitors and their interactions with commonly utilized anesthetic agents is paramount. There are two potentially increased risks related to the continuation of SNRIs through the perioperative period: intraoperative bleeding and serotonin syndrome. SNRIs have some off-label uses, more new indications, and ever-increasing new applications in perioperative practice. This article aims to review the commonly prescribed serotonin-norepinephrine reuptake inhibitors and the current clinical evidence regarding their considerations in perioperative anesthesia and analgesia.

VCP Protects Neurons from Proteopathic Seeding

Background: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), tau, and TDP-43, contribute to neurodegeneration and disease progression. The cellular machinery necessary for this process is poorly understood. Methods: Cas9 expressing αS FRET biosensors were transduced with a whole-genome guide RNA (gRNA) library, seeded with αS fibrils, and flow-sorted. Candidate genes protective against αS seeding were identified following gRNA sequencing of FRET+ and FRET- cell populations. Secondary validation of the high probability candidate suppressor VCP, utilized VCP inhibitors or gene knockdown in αS biosensors and primary neurons. In vivo validation was performed in VCP disease mutation mice following intrastriatal injection of αS seeds. TDP-43 seeding was performed in primary neurons from control or VCP mutant mice.Results: We devised a genome-wide CRISPR-Cas9 screen to identify suppressors of αS seeding. This approach identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) a phenotypically and pathologically variable neurodegeneraive disease characterized by myopathy, motor neuron disease and dementia with TDP-43, αS and tau inclusions. VCP inhibition or MSP disease mutations increased αS seeding in cells and primary cultured neurons. This was similar to treatment with the lysosomal damaging agent, LLoMe or knockdown of the endolysosomal damage response associated VCP cofactor, UBXD1. Intrastriatal injection of αS seeds into VCP disease mice demonstrated enhanced seeding efficiency as compared with controls. Finally, this phenomenon was not specific to αS since VCP disease mutant expression increased TDP-43 seeding in neurons.Conclusion: VCP surveillance of permeabilized late endosomes protects neurons against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Altered Grooming Syntax and Amphetamine-Induced Dopamine Release in EAAT3 Overexpressing Mice

The excitatory amino acid transporter EAAT3 plays an important role in the neuronal uptake of glutamate regulating the activation of glutamate receptors. Polymorphisms in the gene-encoding EAAT3 have been associated with obsessive–compulsive disorder (OCD), although the mechanisms underlying this relationship are still unknown. We recently reported that mice with increased EAAT3 expression in forebrain neurons (EAAT3glo/CMKII) display behavioral and synaptic features relevant to OCD, including increased grooming, higher anxiety-like behavior and altered cortico-striatal synaptic function. The dopamine neurotransmitter system is implicated in ritualistic behaviors. Indeed, dopaminergic neurons express EAAT3, and mice lacking EAAT3 exhibit decreased dopamine release and decreased expression of the dopamine D1 receptor. Moreover, EAAT3 plays a role on the effect of the psychostimulant amphetamine. As such, we sought to determine if the OCD-like behavior in EAAT3glo/CMKII mice is accompanied by altered nigro-striatal dopaminergic transmission. The aim of this study was to analyze dopamine transmission both in basal conditions and after an acute challenge of amphetamine, using behavioral, neurochemical, molecular, and cellular approaches. We found that in basal conditions, EAAT3glo/CMKII mice performed more grooming events and that they remained in phase 1 of the grooming chain syntax compared with control littermates. Administration of amphetamine increased the number of grooming events in control mice, while EAAT3glo/CMKII mice remain unaffected. Interestingly, the grooming syntax of amphetamine-control mice resembled that of EAAT3glo/CMKII mice in basal conditions. Using in vivo microdialysis, we found decreased basal dopamine levels in EAAT3glo/CMKII compared with control mice. Unexpectedly, we found that after acute amphetamine, EAAT3glo/CMKII mice had a higher release of dopamine compared with that of control mice, suggesting that EAAT3 overexpression leads to increased dopamine releasability. To determine postsynaptic effect of EAAT3 overexpression over dopamine transmission, we performed Western blot analysis of dopaminergic proteins and found that EAAT3glo/CMKII mice have higher expression of D2 receptors, suggesting a higher inhibition of the indirect striatal pathway. Together, the data indicate that EAAT3 overexpression impacts on dopamine transmission, making dopamine neurons more sensitive to the effect of amphetamine and leading to a disbalance between the direct and indirect striatal pathways that favors the performance of repetitive behaviors.

ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

Sortilin is a receptor for neuronal uptake of apolipoprotein E. Sortilin-dependent uptake of lipidated apoE promotes conversion of polyunsaturated fatty acids (PUFA) into neuromodulators that induce anti-inflammatory gene expression in the brain. This neuroprotective pathway works with apoE3 but is lost with apoE4, the main risk factor for Alzheimer disease (AD). Here, we elucidated steps in cellular handling of lipids through sortilin, and why they are disrupted by apoE4. Combining unbiased proteome screens with analyses in mouse models, we uncover interaction of sortilin with fatty acid-binding protein (FABP) 7, the intracellular carrier for PUFA in the brain. In the presence of apoE3, sortilin promotes functional expression of FABP7 and its ability to elicit lipid-dependent gene transcription. By contrast, apoE4 binding blocks sortilin sorting, causing catabolism of FABP7 and impairing lipid signaling. Reduced FABP7 levels in the brain of AD patients expressing apoE4 substantiate the relevance of these interactions for neuronal lipid homeostasis. Taken together, we document interaction of sortilin with mediators of extracellular and intracellular lipid transport that provides a mechanistic explanation for loss of a neuroprotective lipid metabolism in AD.

Appraisal of Nano-lipidic Astaxanthin cum Thermoreversible Gel and its Efficacy in Haloperidol Induced Parkinsonism

Background: Parkinsonism has a toxic cascade of neurodegeneration, with akinesia as a major manifestation. Some antioxidants have shown promise against the disease. Astaxanthin is a powerful antioxidant, demonstrates free radical scavenging, and is also a potential neuroprotective agent Objective: To formulate astaxanthin laden nanostructured lipid carriers based thermoreversible gel for better neuronal uptake and better neuronal efficacy. Methods: The method for fabricating astaxanthin-nanostructured lipid carriers (ATX-NLC) was melt-emulsification, and these were optimized using factorial design and further evaluated for diverse parameters. Neurotoxicity was induced in rats by haloperidol. The treated and non-treated rats were then witnessed for their behaviour. TBARs and GSH levels were also determined. Pharmacokinetics was studied via HPLC. Results: The average particle size (by DLS), entrapment efficiency and zeta potential of optimized ATX-NLC were 225.6 ± 3.04 nm, 65.91 ± 1.22 % and -52.64 mV respectively. Astaxanthin release (after 24 h in simulated nasal fluid) from optimized ATX-NLC was 92.5 ± 5.42 %. Its thermo-reversible nasal gel (ATX-NLC in-situ gel) was prepared using poloxamer-127. The obtained gel showed in-vivo betterment in the behaviour of animals when studied using rotarod and akinesia test. Pharmacokinetic studies showed better availability of astaxanthin in the brain on the rats treated with ATX-NLC in-situ gel as compared to those treated with ATX-in-situ gel. Conclusion: Astaxanthin loaded lipidic nanoparticulate gel can be a hopeful adjuvant therapy for Parkinsonism and holds scope for future studies.

Improvement of Negative Symptoms in Schizophrenia: A Doubleblind Clinical Trial by Escitalopram in Male Patients

Background: The negative symptoms of schizophrenia remain a major clinical trouble against psychiatric rehabilitation and available therapeutic treatments. Objective: Escitalopram is known as the most selective SSRI with minimal effects on norepinephrine and dopamine neuronal uptake. The aim of the present study is to assess the effect of escitalopram on negative symptoms of schizophrenia. Methods: This study was an eight-week, randomized, placebo-controlled trial of escitalopram set against placebo, as an add-on medication, in the treatment of 50 patients with a diagnosis of schizophrenia. While the Scale for Assessment of Negative Symptoms was used as the primary outcome measure, the Scale for Assessment of Positive Symptoms, the Simpson-Angus Scale and the Hamilton Depression Scale, as well, were used as a secondary measure for evaluation of positive, extrapyramidal and depressive symptoms, respectively. Results: The primary outcome of the present assessment was a significant reduction in the mean total score of the Scale for Assessment of Negative Symptoms (SANS) in the target group, compared to placebo, at the end of eight weeks. In this regard, most of the subscales of SANS, as well, demonstrated significant improvements by escitalopram. Conclusion: According to the findings, escitalopram can be helpful, as add-on medication, in amelioration of negative symptoms of schizophrenia.

Evidence for aggregation-independent, PrPC-mediated Aβ cellular internalization

Evidence linking amyloid beta (Aβ) cellular uptake and toxicity has burgeoned, and mechanisms underlying this association are subjects of active research. Two major, interconnected questions are whether Aβ uptake is aggregation-dependent and whether it is sequence-specific. We recently reported that the neuronal uptake of Aβ depends significantly on peptide chirality, suggesting that the process is predominantly receptor-mediated. Over the past decade, the cellular prion protein (PrPC) has emerged as an important mediator of Aβ-induced toxicity and of neuronal Aβ internalization. Here, we report that the soluble, nonfibrillizing Aβ (1–30) peptide recapitulates full-length Aβ stereoselective cellular uptake, allowing us to decouple aggregation from cellular, receptor-mediated internalization. Moreover, we found that Aβ (1–30) uptake is also dependent on PrPCexpression. NMR-based molecular-level characterization identified the docking site on PrPCthat underlies the stereoselective binding of Aβ (1–30). Our findings therefore identify a specific sequence within Aβ that is responsible for the recognition of the peptide by PrPC, as well as PrPC-dependent cellular uptake. Further uptake stereodifferentiation in PrPC-free cells points toward additional receptor-mediated interactions as likely contributors for Aβ cellular internalization. Taken together, our results highlight the potential of targeting cellular surface receptors to inhibit Aβ cellular uptake as an alternative route for future therapeutic development for Alzheimer’s disease.

Whole genome CRISPR screens identify LRRK2-regulated endocytosis as a major mechanism for extracellular tau uptake by human neurons

ABSTRACTPathological protein aggregation in Alzheimer’s disease and other dementias is proposed to spread through the nervous system by a process of intercellular transfer of pathogenic forms of tau protein. Defining the cellular mechanisms of tau entry to human neurons is essential for understanding dementia pathogenesis and the rational design of disease-modifying therapeutics. Using whole genome CRISPR knockout screens in human iPSC-derived excitatory neurons, the primary cell type affected in these diseases, we identified genes and pathways required specifically for uptake of monomeric and aggregated tau. Monomeric and aggregated tau are both taken up by human neurons by receptor-mediated endocytosis, with the low-density lipoprotein LRP1 a significant surface receptor for both forms of tau. Perturbations of the endolysosome and autophagy systems at many levels, and specifically endosome sorting and receptor recycling, greatly reduced tau uptake. Of particular therapeutic interest is that loss of function of the endocytosis and autophagy regulator LRRK2, as well as acute inhibition of its kinase activity, reduced neuronal uptake of monomeric and aggregated tau. Kinase-activating mutations in LRRK2 are a cause of Parkinson’s disease accompanied by neuronal tau aggregation, suggesting that LRRK2 mediates tau spreading in vivo and that LRRK2 inhibition has the potential to inhibit interneuronal spread of tau pathology, slowing disease progression. Overall, pathways for tau entry share significant similarity with those required for virus entry by receptor-mediated endocytosis, suggesting that tau spreading is a quasi-infectious process.