Glutamate and GABA in Microglia-Neuron Cross-Talk in Alzheimer’s Disease

The physiological balance between excitation and inhibition in the brain is significantly affected in Alzheimer’s disease (AD). Several neuroactive compounds and their signaling pathways through various types of receptors are crucial in brain homeostasis, among them glutamate and γ-aminobutyric acid (GABA). Activation of microglial receptors regulates the immunological response of these cells, which in AD could be neuroprotective or neurotoxic. The novel research approaches revealed the complexity of microglial function, including the interplay with other cells during neuroinflammation and in the AD brain. The purpose of this review is to describe the role of several proteins and multiple receptors on microglia and neurons, and their involvement in a communication network between cells that could lead to different metabolic loops and cell death/survival. Our review is focused on the role of glutamatergic, GABAergic signaling in microglia–neuronal cross-talk in AD and neuroinflammation. Moreover, the significance of AD-related neurotoxic proteins in glutamate/GABA-mediated dialogue between microglia and neurons was analyzed in search of novel targets in neuroprotection, and advanced pharmacological approaches.     

Neuroprotective and Anti-Inflammatory Effects of Evernic Acid in an MPTP-Induced Parkinson’s Disease Model

Oxidative stress, mitochondrial dysfunction, and neuroinflammation are strongly associated with the pathogenesis of Parkinson’s disease (PD), which suggests that anti-oxidative and anti-inflammatory compounds might provide an alternative treatment for PD. Here, we evaluated the neuroprotective effects of evernic aid (EA), which was screened from a lichen library provided by the Korean Lichen Research Institute at Sunchon National University. EA is a secondary metabolite generated by lichens, including Ramalina, Evernia, and Hypogymnia, and several studies have described its anticancer, antifungal, and antimicrobial effects. However, the neuroprotective effects of EA have not been studied. We found that EA protected primary cultured neurons against 1-methyl-4-phenylpyridium (MPP⁺)-induced cell death, mitochondrial dysfunction, and oxidative stress, and effectively reduced MPP⁺-induced astroglial activation by inhibiting the NF-κB pathway. In vivo, EA ameliorated MPTP-induced motor dysfunction, dopaminergic neuronal loss, and neuroinflammation in the nigrostriatal pathway in C57BL/6 mice. Taken together, our findings demonstrate that EA has neuroprotective and anti-inflammatory effects in PD models and suggest that EA is a potential therapeutic candidate for PD.     

A Possible Role for HMG-CoA Reductase Inhibitors and Its Association with HMGCR Genetic Variation in Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease characterised by both motor- and non-motor symptoms, including cognitive impairment. The aetiopathogenesis of PD, as well as its protective and susceptibility factors, are still elusive. Neuroprotective effects of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors—statins—via both cholesterol-dependent and independent mechanisms have been shown in animal and cell culture models. However, the available data provide conflicting results on the role of statin treatment in PD patients. Moreover, cholesterol is a vital component for brain functions and may be considered as protective against PD. We present possible statin effects on PD under the hypothesis that they may depend on the HMG-CoA reductase gene (HMGCR) variability, such as haplotype 7, which was shown to affect cholesterol synthesis and statin treatment outcome, diminishing possible neuroprotection associated with HMG-CoA reductase inhibitors administration. Statins are among the most prescribed groups of drugs. Thus, it seems important to review the available data in the context of their possible neuroprotective effects in PD, and the HMG-CoA reductase gene’s genetic variability.     

New Insights into Immune-Mediated Mechanisms in Parkinson’s Disease

The immune system has been increasingly recognized as a major contributor in the pathogenesis of Parkinson’s disease (PD). The double-edged nature of the immune system poses a problem in harnessing immunomodulatory therapies to prevent and slow the progression of this debilitating disease. To tackle this conundrum, understanding the mechanisms underlying immune-mediated neuronal death will aid in the identification of neuroprotective strategies to preserve dopaminergic neurons. Specific innate and adaptive immune mediators may directly or indirectly induce dopaminergic neuronal death. Genetic factors, the gut-brain axis and the recent identification of PD-specific T cells may provide novel mechanistic insights on PD pathogenesis. Future studies to address the gaps in the identification of autoantibodies, variability in immunophenotyping studies and the contribution of gut dysbiosis to PD may eventually provide new therapeutic targets for PD.     

Neurodegeneration and Inflammation—An Interesting Interplay in Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder, caused by, so far, unknown pathogenetic mechanisms. There is no doubt that pro-inflammatory immune-mediated mechanisms are pivotal to the pathogenicity and progression of the disease. In this review, we highlight the binary role of microglia activation in the pathophysiology of the disorder, both neuroprotective and neuromodulatory. We present how the expression of several cytokines implicated in dopaminergic neurons (DA) degeneration could be used as biomarkers for PD. Viral infections have been studied and correlated to the disease progression, usually operating as trigger factors for the inflammatory process. The gut–brain axis and the possible contribution of the peripheral bowel inflammation to neuronal death, mainly dopaminergic neurons, seems to be a main contributor of brain neuroinflammation. The role of the immune system has also been analyzed implicating a-synuclein in the activation of innate and adaptive immunity. We also discuss therapeutic approaches concerning PD and neuroinflammation, which have been studied in experimental and in vitro models and data stemming from epidemiological studies.     

In Vitro and in Vivo Neuroprotective Effects of Walnut (Juglandis Semen) in Models of Parkinson’s Disease

Monoamine oxidase (MAO) catalyzes the oxidative deamination of monoamines including dopamine (DA). MAO expression is elevated in Parkinson’s disease (PD). An increase in MAO activity is closely related to age, and this may induce neuronal degeneration in the brain due to oxidative stress. MAO (and particularly monoamine oxidase B (MAO-B)) participates in the generation of reactive oxygen species (ROS), such as hydrogen peroxide that are toxic to dopaminergic cells and their surroundings. Although the polyphenol-rich aqueous walnut extract (JSE; an extract of Juglandis Semen) has been shown to have various beneficial bioactivities, no study has been dedicated to see if JSE is capable to protect dopaminergic neurons against neurotoxic insults in models of PD. In the present study we investigated the neuroprotective potential of JSE against 1-methyl-4-phenylpyridinium (MPP⁺)- or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicities in primary mesencephalic cells and in a mouse model of PD. Here we show that JSE treatment suppressed ROS and nitric oxide productions triggered by MPP⁺ in primary mesencephalic cells. JSE also inhibited depletion of striatal DA and its metabolites in vivo that resulted in significant improvement in PD-like movement impairment. Altogether our results indicate that JSE has neuroprotective effects in PD models and may have potential for the prevention or treatment of PD.     

Epigenetic Regulation of Neuroinflammation in Parkinson’s Disease

Neuroinflammation is one of the most significant factors involved in the initiation and progression of Parkinson’s disease. PD is a neurodegenerative disorder with a motor disability linked with various complex and diversified risk factors. These factors trigger myriads of cellular and molecular processes, such as misfolding defective proteins, oxidative stress, mitochondrial dysfunction, and neurotoxic substances that induce selective neurodegeneration of dopamine neurons. This neuronal damage activates the neuronal immune system, including glial cells and inflammatory cytokines, to trigger neuroinflammation. The transition of acute to chronic neuroinflammation enhances the susceptibility of inflammation-induced dopaminergic neuron damage, forming a vicious cycle and prompting an individual to PD development. Epigenetic mechanisms recently have been at the forefront of the regulation of neuroinflammatory factors in PD, proposing a new dawn for breaking this vicious cycle. This review examined the core epigenetic mechanisms involved in the activation and phenotypic transformation of glial cells mediated neuroinflammation in PD. We found that epigenetic mechanisms do not work independently, despite being coordinated with each other to activate neuroinflammatory pathways. In this regard, we attempted to find the synergic correlation and contribution of these epigenetic modifications with various neuroinflammatory pathways to broaden the canvas of underlying pathological mechanisms involved in PD development. Moreover, this study highlighted the dual characteristics (neuroprotective/neurotoxic) of these epigenetic marks, which may counteract PD pathogenesis and make them potential candidates for devising future PD diagnosis and treatment.     

Parkinson’s Disease-Related Genes and Lipid Alteration

Parkinson’s disease (PD) is a complex and progressive neurodegenerative disorder with a prevalence of approximately 0.5–1% among those aged 65–70 years. Although most of its clinical manifestations are due to a loss of dopaminergic neurons, the PD etiology is largely unknown. PD is caused by a combination of genetic and environmental factors, and the exact interplay between genes and the environment is still debated. Several biological processes have been implicated in PD, including mitochondrial or lysosomal dysfunctions, alteration in protein clearance, and neuroinflammation, but a common molecular mechanism connecting the different cellular alterations remains incompletely understood. Accumulating evidence underlines a significant role of lipids in the pathological pathways leading to PD. Beside the well-described lipid alteration in idiopathic PD, this review summarizes the several lipid alterations observed in experimental models expressing PD-related genes and suggests a possible scenario in relationship to the molecular mechanisms of neuronal toxicity. PD could be considered a lipid-induced proteinopathy, where alteration in lipid composition or metabolism could induce protein alteration—for instance, alpha-synuclein accumulation—and finally neuronal death.     

Modeling Parkinson’s Disease Neuropathology and Symptoms by Intranigral Inoculation of Preformed Human α-Synuclein Oligomers

The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson’s disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies.     

Therapeutic Potential of AAV1-Rheb(S16H) Transduction against Neurodegenerative Diseases

Neurotrophic factors (NTFs) are essential for cell growth, survival, synaptic plasticity, and maintenance of specific neuronal population in the central nervous system. Multiple studies have demonstrated that alterations in the levels and activities of NTFs are related to the pathology and symptoms of neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease. Hence, the key molecule that can regulate the expression of NTFs is an important target for gene therapy coupling adeno-associated virus vector (AAV) gene. We have previously reported that the Ras homolog protein enriched in brain (Rheb)–mammalian target of rapamycin complex 1 (mTORC1) axis plays a vital role in preventing neuronal death in the brain of AD and PD patients. AAV transduction using a constitutively active form of Rheb exerts a neuroprotective effect through the upregulation of NTFs, thereby promoting the neurotrophic interaction between astrocytes and neurons in AD conditions. These findings suggest the role of Rheb as an important regulator of the regulatory system of NTFs to treat neurodegenerative diseases. In this review, we present an overview of the role of Rheb in neurodegenerative diseases and summarize the therapeutic potential of AAV serotype 1 (AAV1)-Rheb(S16H) transduction in the treatment of neurodegenerative disorders, focusing on diseases, such as AD and PD.     

The Contribution of Microglia to Neuroinflammation in Parkinson’s Disease

With the world’s population ageing, the incidence of Parkinson’s disease (PD) is on the rise. In recent years, inflammatory processes have emerged as prominent contributors to the pathology of PD. There is great evidence that microglia have a significant neuroprotective role, and that impaired and over activated microglial phenotypes are present in brains of PD patients. Thereby, PD progression is potentially driven by a vicious cycle between dying neurons and microglia through the instigation of oxidative stress, mitophagy and autophagy dysfunctions, a-synuclein accumulation, and pro-inflammatory cytokine release. Hence, investigating the involvement of microglia is of great importance for future research and treatment of PD. The purpose of this review is to highlight recent findings concerning the microglia-neuronal interplay in PD with a focus on human postmortem immunohistochemistry and single-cell studies, their relation to animal and iPSC-derived models, newly emerging technologies, and the resulting potential of new anti-inflammatory therapies for PD.     

Transthyretin Stabilization: An Emerging Strategy for the Treatment of Alzheimer’s Disease?

Transthyretin (TTR), previously named prealbumin is a plasma protein secreted mainly by the liver and choroid plexus (CP) that is a carrier for thyroid hormones (THs) and retinol (vitamin A). The structure of TTR, with four monomers rich in β-chains in a globular tetrameric protein, accounts for the predisposition of the protein to aggregate in fibrils, leading to a rare and severe disease, namely transthyretin amyloidosis (ATTR). Much effort has been made and still is required to find new therapeutic compounds that can stabilize TTR (“kinetic stabilization”) and prevent the amyloid genetic process. Moreover, TTR is an interesting therapeutic target for neurodegenerative diseases due to its recognized neuroprotective properties in the cognitive impairment context and interestingly in Alzheimer’s disease (AD). Much evidence has been collected regarding the neuroprotective effects in AD, including through in vitro and in vivo studies as well as a wide range of clinical series. Despite this supported hypothesis of neuroprotection for TTR, the mechanisms are still not completely clear. The aim of this review is to highlight the most relevant findings on the neuroprotective role of TTR, and to summarize the recent progress on the development of TTR tetramer stabilizers.     

Oleuropein Prevents Neuronal Death,Mitigates Mitochondrial Superoxide Production and Modulates Autophagy in a Dopaminergic Cellular Model

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, primarily affecting dopaminergic neurons in the substantia nigra. There is currently no cure for PD and present medications aim to alleviate clinical symptoms, thus prevention remains the ideal strategy to reduce the prevalence of this disease. The goal of this study was to investigate whether oleuropein (OLE), the major phenolic compound in olive derivatives, may prevent neuronal degeneration in a cellular dopaminergic model of PD, differentiated PC12 cells exposed to the potent parkinsonian toxin 6-hydroxydopamine (6-OHDA). We also investigated OLE’s ability to mitigate mitochondrial oxidative stress and modulate the autophagic flux. Our results obtained by measuring cytotoxicity and apoptotic events demonstrate that OLE significantly decreases neuronal death. OLE could also reduce mitochondrial production of reactive oxygen species resulting from blocking superoxide dismutase activity. Moreover, quantification of autophagic and acidic vesicles in the cytoplasm alongside expression of specific autophagic markers uncovered a regulatory role for OLE against autophagic flux impairment induced by bafilomycin A1. Altogether, our results define OLE as a neuroprotective, anti-oxidative and autophagy-regulating molecule, in a neuronal dopaminergic cellular model.     

Synaptic Zinc: An Emerging Player in Parkinson’s Disease

Alterations of zinc homeostasis have long been implicated in Parkinson’s disease (PD). Zinc plays a complex role as both deficiency and excess of intracellular zinc levels have been incriminated in the pathophysiology of the disease. Besides its role in multiple cellular functions, Zn²⁺ also acts as a synaptic transmitter in the brain. In the forebrain, subset of glutamatergic neurons, namely cortical neurons projecting to the striatum, use Zn²⁺ as a messenger alongside glutamate. Overactivation of the cortico-striatal glutamatergic system is a key feature contributing to the development of PD symptoms and dopaminergic neurotoxicity. Here, we will cover recent evidence implicating synaptic Zn²⁺ in the pathophysiology of PD and discuss its potential mechanisms of actions. Emphasis will be placed on the functional interaction between Zn²⁺ and glutamatergic NMDA receptors, the most extensively studied synaptic target of Zn²⁺.     

A Novel LRRK2 Variant p.G2294R in the WD40 Domain Identified in Familial Parkinson’s Disease Affects LRRK2 Protein Levels

Leucine-rich repeat kinase 2 (LRRK2) is a major causative gene of late-onset familial Parkinson’s disease (PD). The suppression of kinase activity is believed to confer neuroprotection, as most pathogenic variants of LRRK2 associated with PD exhibit increased kinase activity. We herein report a novel LRRK2 variant—p.G2294R—located in the WD40 domain, detected through targeted gene-panel screening in a patient with familial PD. The proband showed late-onset Parkinsonism with dysautonomia and a good response to levodopa, without cognitive decline or psychosis. Cultured cell experiments revealed that p.G2294R is highly destabilized at the protein level. The LRRK2 p.G2294R protein expression was upregulated in the patient’s peripheral blood lymphocytes. However, macrophages differentiated from the same peripheral blood showed decreased LRRK2 protein levels. Moreover, our experiment indicated reduced phagocytic activity in the pathogenic yeasts and α-synuclein fibrils. This PD case presents an example wherein the decrease in LRRK2 activity did not act in a neuroprotective manner. Further investigations are needed in order to elucidate the relationship between LRRK2 expression in the central nervous system and the pathogenesis caused by altered LRRK2 activity.     

The Involvement of Insulin-Like Growth Factor 1 and Nerve Growth Factor in Alzheimer’s Disease-Like Pathology and Survival Role of the Mix of Embryonic Proteoglycans: Electrophysiological Fingerprint,Structural Changes and Regulatory Effects on Neurotrophins

Alzheimer’s disease (AD)-associated neurodegeneration is triggered by different fragments of amyloid beta (Aβ). Among them, Aβ (25–35) fragment plays a critical role in the development of neurodegeneration—it reduces synaptic integrity by disruption of excitatory/inhibitory ratio across networks and alters the growth factors synthesis. Thus, in this study, we aimed to identify the involvement of neurotrophic factors—the insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF)—of AD-like neurodegeneration induced by Aβ (25–35). Taking into account our previous findings on the neuroprotective effects of the mix of proteoglycans of embryonic genesis (PEG), it was suggested to test its regulatory effect on IGF-1 and NGF levels. To evaluate the progress of neurodegeneration, in vivo electrophysiological investigation of synaptic activity disruption of the entorhinal cortex–hippocampus circuit at AD was performed and the potential recovery effects of PEG with relative structural changes were provided. To reveal the direct effects of PEG on brain functional activity, the electrophysiological pattern of the single cells from nucleus supraopticus, sensomotor cortex and hippocampus after acute injection of PEG was examined. Our results demonstrated that after i.c.v. injection of Aβ (25–35), the level of NGF decreased in cerebral cortex and hypothalamus, and, in contrast, increased in hippocampus, prompting its multidirectional role in case of brain damage. The concentration of IGF-1 significantly increased in all investigated brain structures. The administration of PEG balanced the growth factor levels accompanied by substantial restoration of neural tissue architecture and synaptic activity. Acute injection of PEG activated the hypothalamic nucleus supraopticus and hippocampal neurons. IGF-1 and NGF levels were found to be elevated in animals receiving PEG in an absence of amyloid exposure. We suggest that IGF-1 and NGF play a critical role in the development of AD. At the same time, it becomes clear that the neuroprotective effects of PEG are likely mediated via the regulation of neurotrophins.     

Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.     

A Role for Xanthurenic Acid in the Control of Brain Dopaminergic Activity

Xanthurenic acid (XA) is a metabolite of the kynurenine pathway (KP) synthetized in the brain from dietary or microbial tryptophan that crosses the blood-brain barrier through carrier-mediated transport. XA and kynurenic acid (KYNA) are two structurally related compounds of KP occurring at micromolar concentrations in the CNS and suspected to modulate some pathophysiological mechanisms of neuropsychiatric and/or neurodegenerative diseases. Particularly, various data including XA cerebral distribution (from 1 µM in olfactory bulbs and cerebellum to 0.1–0.4 µM in A₉ and A₁₀), its release, and interactions with G protein-dependent XA-receptor, glutamate transporter and metabotropic receptors, strongly support a signaling and/or neuromodulatory role for XA. However, while the parent molecule KYNA is considered as potentially involved in neuropsychiatric disorders because of its inhibitory action on dopamine release in the striatum, the effect of XA on brain dopaminergic activity remains unknown. Here, we demonstrate that acute local/microdialysis-infusions of XA dose-dependently stimulate dopamine release in the rat prefrontal cortex (four-fold increase in the presence of 20 µM XA). This stimulatory effect is blocked by XA-receptor antagonist NCS-486. Interestingly, our results show that the peripheral/intraperitoneal administration of XA, which has been proven to enhance intra-cerebral XA concentrations (about 200% increase after 50 mg/kg XA i.p), also induces a dose-dependent increase of dopamine release in the cortex and striatum. Furthermore, our in vivo electrophysiological studies reveal that the repeated/daily administrations of XA reduce by 43% the number of spontaneously firing dopaminergic neurons in the ventral tegmental area. In the substantia nigra, XA treatment does not change the number of firing neurons. Altogether, our results suggest that XA may contribute together with KYNA to generate a KYNA/XA ratio that may crucially determine the brain normal dopaminergic activity. Imbalance of this ratio may result in dopaminergic dysfunctions related to several brain disorders, including psychotic diseases and drug dependence.     

A Pilot Study of Changes in the Level of Catecholamines and the Activity of α-2-Macroglobulin in the Tear Fluid of Patients with Parkinson’s Disease and Parkinsonian Mice

Development of differential and early (preclinical) diagnostics of Parkinson’s disease (PD) is among the priorities in neuroscience. We searched for changes in the level of catecholamines and α-2-macroglobulin activity in the tear fluid (TF) in PD patients at an early clinical stage. It was shown that TF in patients is characterized by an increased level of noradrenaline mainly on the ipsilateral side of pronounced motor symptoms (72%, p = 0.049), a decreased level of adrenaline on both sides (ipsilateral—53%, p = 0.004; contralateral—42%, p = 0.02), and an increased α-2-macroglobulin activity on both sides (ipsilateral—53%, p = 0.03; contralateral—56%, p = 0.037) compared to controls. These changes are considered as potential biomarkers for differential diagnosis. Similar changes in the TF were found in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice when modeling clinical and preclinical stages of PD. These data show the adequacy of models to the pathogenesis of PD along the selected metabolic pathways, and also suggest that the found TF changes can be considered as potential biomarkers for preclinical diagnosis of PD. In Parkinsonian mice, the level of catecholamines also changes in the lacrimal glands, which makes it possible to consider them as one of the sources of catecholamines in the TF.