Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119+CD45-CD71+ phenotype. Ter-cells are enriched in the enlarged spleen of hosts bearing advanced tumors and facilitate tumor progression by secreting neurotrophic factor artemin into the blood. Transforming growth factor ß (TGF-ß) and Smad3 activation are important in Ter-cell generation. In vivo blockade of Ter-cell-derived artemin inhibits hepatocellular carcinoma (HCC) growth, and artemin deficiency abolishes Ter-cells' tumor-promoting ability. We confirm the presence of splenic artemin-positive Ter-cells in human HCC patients and show that significantly elevated serum artemin correlates with poor prognosis. We propose that Ter-cells and the secreted artemin play important roles in cancer progression with prognostic and therapeutic implications.

Diverse immunological dysregulation, chronic inflammation, and impaired erythropoiesis in long COVID patients with chronic fatigue syndrome.

A substantial number of patients recovering from acute SARS-CoV-2 infection present serious lingering symptoms, often referred to as long COVID (LC). However, a subset of these patients exhibits the most debilitating symptoms characterized by ongoing myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS). We specifically identified and studied ME/CFS patients from two independent LC cohorts, at least 12 months post the onset of acute disease, and compared them to the recovered group (R). ME/CFS patients had relatively increased neutrophils and monocytes but reduced lymphocytes. Selective T cell exhaustion with reduced naïve but increased terminal effector T cells was observed in these patients. LC was associated with elevated levels of plasma pro-inflammatory cytokines, chemokines, Galectin-9 (Gal-9), and artemin (ARTN). A defined threshold of Gal-9 and ARTN concentrations had a strong association with LC. The expansion of immunosuppressive CD71+ erythroid cells (CECs) was noted. These cells may modulate the immune response and contribute to increased ARTN concentration, which correlated with pain and cognitive impairment. Serology revealed an elevation in a variety of autoantibodies in LC. Intriguingly, we found that the frequency of 2B4+CD160+ and TIM3+CD160+ CD8+ T cells completely separated LC patients from the R group. Our further analyses using a multiple regression model revealed that the elevated frequency/levels of CD4 terminal effector, ARTN, CEC, Gal-9, CD8 terminal effector, and MCP1 but lower frequency/levels of TGF-ß and MAIT cells can distinguish LC from the R group. Our findings provide a new paradigm in the pathogenesis of ME/CFS to identify strategies for its prevention and treatment.

Artemin sensitizes nociceptors that innervate the osteoarthritic joint to produce pain.

OBJECTIVE: There have been significant developments in understanding artemin/GFRα3 signaling in recent years, and there is now accumulating evidence that artemin has important roles to play in pain signaling, including that derived from joint and bone, and that associated with osteorthritis (OA). METHODS: A total of 163 Sprague-Dawley rats were used in this study. We used an animal model of mono-iodoacetate (MIA)-induced OA, in combination with electrophysiology, behavioral testing, Western blot analysis, and retrograde tracing and immunohistochemistry, to identify roles for artemin/GFRα3 signaling in the pathogenesis of OA pain. RESULTS: We have found that: 1) GFRα3 is expressed in a substantial proportion of knee joint afferent neurons; 2) exogenous artemin sensitizes knee joint afferent neurons in naïve rats; 3) artemin is expressed in articular tissues of the joint, but not surrounding bone, early in MIA-induced OA; 4) artemin expression increases in bone later in MIA-induced OA when pathology involves subchondral bone; and 5) sequestration of artemin reverses MIA-induced sensitization of both knee joint and bone afferent neurons late in disease when there is inflammation of knee joint tissues and damage to the subchondral bone. CONCLUSIONS: Our findings show that artemin/GFRα3 signaling has a role to play in the pathogenesis of OA pain, through effects on both knee joint and bone afferent neurons, and suggest that targeted manipulation of artemin/GFRα3 signaling may provide therapeutic benefit for the management of OA pain. DATA AVAILABILITY: Data are available on request of the corresponding author.

Human splenic TER cells: A relevant prognostic factor acting via the artemin-GFRα3-ERK pathway in pancreatic ductal adenocarcinoma.

Splenectomy is routinely performed during distal or total pancreatectomy (DP or TP) for pancreatic ductal adenocarcinoma (PDAC), but information about its oncological value is limited. TER cells, nonimmune cells discovered in the spleens of tumour-bearing mice, are elicited by tumours and promote tumour progression, while their role in the clinical outcomes of patients with PDAC remains unclear. In our study, postoperative specimens from 622 patients who underwent DP or TP with splenectomy were analysed by flow cytometry or immunofluorescence, and the relationship between splenic TER cell count and clinical parameters was calculated. We also purified human TER cells for functional experiments and mechanistic studies. We found that TER cell numbers were increased only in the spleens of patients with PDAC but not in PDAC tissue and adjacent pancreatic tissue. High splenic TER cell counts independently predicted poor prognosis (P < .001) and indicated large tumour size, lymph node metastasis, advanced 8th AJCC/mAJCC stage and high CA19-9 classification (all P < .050) in patients with PDAC. Mechanistic analysis showed that TER cells express artemin, which facilitates the proliferation and invasion of PDAC cells by activating GFRα3-ERK signalling. Our study reveals that TER cell count is an indicator of poor prognosis of PDAC, while splenectomy during pancreatic surgery might provide oncological benefits in addition to ensuring the radical resection of PDAC.

Novel RET agonist for the treatment of experimental neuropathies.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) alleviate symptoms of experimental neuropathy, protect and stimulate regeneration of sensory neurons in animal models of neuropathic pain, and restore their functional activity. However, clinical development of GFL proteins is complicated by their poor pharmacokinetic properties and multiple effects mediated by several receptors. Previously, we have identified a small molecule that selectively activates the major signal transduction unit of the GFL receptor complex, receptor tyrosine kinase RET, as an alternative to GFLs, for the treatment of neuropathic pain. We then introduced a series of chemical changes to improve the biological activity of these compounds and tested an optimized compound named BT44 in a panel of biological assays. BT44 efficiently and selectively stimulated the GFL receptor RET and activated the intracellular mitogene-activated protein kinase/extracellular signal-regulated kinase pathway in immortalized cells. In cultured sensory neurons, BT44 stimulated neurite outgrowth with an efficacy comparable to that of GFLs. BT44 alleviated mechanical hypersensitivity in surgery- and diabetes-induced rat models of neuropathic pain. In addition, BT44 normalized, to a certain degree, the expression of nociception-related neuronal markers which were altered by spinal nerve ligation, the neuropathy model used in this study. Our results suggest that the GFL mimetic BT44 is a promising new lead for the development of novel disease-modifying agents for the treatment of neuropathy and neuropathic pain.

Anti-amyloidogenic effect of artemin on α-synuclein.

α-Synuclein fibrillation is now regarded as a major pathogenic process in Parkinson's disease and its proteinaceous deposits are also detected in other neurological disorders including Alzheimer's disease. Therefore anti-amyloidegenic compounds may delay or prevent the progression of synucleinopathies disease. Molecular chaperones are group of proteins which mediate correct folding of proteins by preventing unsuitable interactions which may lead to aggregation. The objective of this study was to investigate the anti-amyloidogenic effect of molecular chaperone artemin on α-synuclein. As the concentration of artemin was increased up to 4 µg/ml, a decrease in fibril formation of α-synuclein was observed using thioflavin T (ThT) fluorescence and congo red (CR) assay. Transmission electron microscopy (TEM) images also demonstrated a reduction in fibrils in the presence of artemin. The secondary structure of α-synuclein was similar to its native form prior to fibrillation when incubated with artemin. A cell-based assay has shown that artemin inhibits α-synuclein aggregation and reduce cytotoxicity, apoptosis and reactive oxygen species (ROS) production. Our results revealed that artemin has efficient chaperon activity for preventing α-synuclein fibril formation and toxicity.

Glial cell line-derived neurotrophic factors (GFLs) and small molecules targeting RET receptor for the treatment of pain and Parkinson's disease.

Rearranged during transfection (RET), in complex with glial cell line-derived (GDNF) family receptor alpha (GFRα), is the canonical signaling receptor for GDNF family ligands (GFLs) expressed in both central and peripheral parts of the nervous system and also in non-neuronal tissues. RET-dependent signaling elicited by GFLs has an important role in the development, maintenance and survival of dopamine and sensory neurons. Both Parkinson's disease and neuropathic pain are devastating disorders without an available cure, and at the moment are only treated symptomatically. GFLs have been studied extensively in animal models of Parkinson's disease and neuropathic pain with remarkable outcomes. However, clinical trials with recombinant or viral vector-encoded GFL proteins have produced inconclusive results. GFL proteins are not drug-like; they have poor pharmacokinetic properties and activate multiple receptors. Targeting RET and/or GFRα with small molecules may resolve the problems associated with using GFLs as drugs and can result in the development of therapeutics for disease-modifying treatments against Parkinson's disease and neuropathic pain.

Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.

Numerous neurotrophic factors promote the survival of developing motor neurons but their combinatorial actions remain poorly understood; to address this, we here screened 66 combinations of 12 neurotrophic factors on pure, highly viable, and standardized embryonic mouse motor neurons isolated by a unique FACS technique. We demonstrate potent, strictly additive, survival effects of hepatocyte growth factor (HGF), ciliary neurotrophic factor (CNTF), and Artemin through specific activation of their receptor complexes in distinct subsets of lumbar motor neurons: HGF supports hindlimb motor neurons through c-Met; CNTF supports subsets of axial motor neurons through CNTFRα; and Artemin acts as the first survival factor for parasympathetic preganglionic motor neurons through GFRα3/Syndecan-3 activation. These data show that neurotrophic factors can selectively promote the survival of distinct classes of embryonic motor neurons. Similar studies on postnatal motor neurons may provide a conceptual framework for the combined therapeutic use of neurotrophic factors in degenerative motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, and spinobulbar muscular atrophy.

Bacopa monnieri as augmentation therapy in the treatment of anhedonia, preclinical and clinical evaluation.

Bacopa monnieri (L.) is widely used in Ayurvedic medicine as a neural tonic for improving intelligence and memory. Several studies highlighted its efficacy in neuropsychiatric diseases but there is no evidence regarding anhedonia. Aim of the present work was to preclinically and clinically test against anhedonia a standardized B. monnieri extract (20% bacosides). In a mouse model of a depressive-like syndrome induced by lipopolysaccharide (LPS), the daily administration of the extract (50-200 mg kg-1 , p.o.) for 1 week, dose-dependently counteracted the immobility time in Porsolt and Tail suspension tests (p < .01). At the sucrose preference test (directly related to the ability for feeling pleasure) the extract treatment (100 and 200 mg kg-1 ) counteracted the reduction of sucrose intake induced by LPS (p < .01). Moreover, B. monnieri significantly reduced cytokines, cortisol, and artemin LPS-dependent alterations in plasma while increased the brain-derived neurotrophic factor levels (p < .05). The efficacy of the same extract was tested in a clinical study in which 42 patients with significant degree of anhedonia (evaluated as Snaith-Hamilton Pleasure Scale [SHAPS] score ≥ 3) were enrolled. Patients were divided into two groups and treated with citalopram or citalopram associated with B. monnieri (300 mg bid) for 4 weeks. The Pears Sample T-test showed a significant improvement (p < .05) in relevant scales (Hamilton depression rating scale, SHAPS, and strength and difficulties questionnaire) in the extract-treated group in comparison to citalopram alone was recorded. These data suggest that B. monnieri extract may be effective for the management of anhedonia and therefore should be considered for future controlled trials.

GDNF, Neurturin, and Artemin Activate and Sensitize Bone Afferent Neurons and Contribute to Inflammatory Bone Pain.

Pain associated with skeletal pathology or disease is a significant clinical problem, but the mechanisms that generate and/or maintain it remain poorly understood. In this study, we explored roles for GDNF, neurturin, and artemin signaling in bone pain using male Sprague Dawley rats. We have shown that inflammatory bone pain involves activation and sensitization of peptidergic, NGF-sensitive neurons via artemin/GDNF family receptor α-3 (GFRα3) signaling pathways, and that sequestering artemin might be useful to prevent inflammatory bone pain derived from activation of NGF-sensitive bone afferent neurons. In addition, we have shown that inflammatory bone pain also involves activation and sensitization of nonpeptidergic neurons via GDNF/GFRα1 and neurturin/GFRα2 signaling pathways, and that sequestration of neurturin, but not GDNF, might be useful to treat inflammatory bone pain derived from activation of nonpeptidergic bone afferent neurons. Our findings suggest that GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.SIGNIFICANCE STATEMENT Pain associated with skeletal pathology, including bone cancer, bone marrow edema syndromes, osteomyelitis, osteoarthritis, and fractures causes a major burden (both in terms of quality of life and cost) on individuals and health care systems worldwide. We have shown the first evidence of a role for GDNF, neurturin, and artemin in the activation and sensitization of bone afferent neurons, and that sequestering these ligands reduces pain behavior in a model of inflammatory bone pain. Thus, GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.

A population of nonneuronal GFRα3-expressing cells in the bone marrow resembles nonmyelinating Schwann cells.

Artemin is a neurotrophic factor that plays a crucial role in the regulation of neural development and regeneration and has also been implicated in the pathogenesis of inflammatory pain. The receptor for artemin, GFRα3, is expressed by sympathetic and nociceptive sensory neurons, including some that innervate the bone marrow, but it is unclear if it is also expressed in other cell types in the bone marrow. Our goal in the present study was to characterise the expression of GFRα3 in nonneuronal cells in the bone marrow. Immunohistochemical studies revealed that GFRα3-expressing cells in the bone marrow are spatially associated with blood vessels and are in intimate contact with nerve fibres. We used various combinations of markers to distinguish different cell types and found that the GFRα3-expressing cells expressed markers of nonmyelinating Schwann cells (e.g. GFAP, p75NTR, nestin). Analysis of bone marrow sections of Wnt1-reporter mice also demonstrated that they originate from the neural crest. Further characterisation using flow cytometry revealed that GFRα3 is expressed in a population of CD51+Sca1-PDGFRα- cells, reinforcing the notion that they are neural crest-derived, nonmyelinating Schwann cells. In conclusion, there is a close association between peripheral nerve terminals and a population of nonneuronal cells that express GFRα3 in the bone marrow. The nonneuronal cells have characteristics consistent with a neural crest-derived, nonmyelinating Schwann cell phenotype. Our findings provide a better understanding of the expression pattern of GFRα3 in the bone marrow microenvironment.

Artemin regulates CXCR4 expression to induce migration and invasion in pancreatic cancer cells through activation of NF-κB signaling.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignant tumor because of the early onset of local invasion and distant metastasis. Perineural invasion is a prominent characteristic of pancreatic adenocarcinoma, which is a multifactorial process that involves various signaling molecules from different signaling pathways. The glial cell line-derived neurotrophic factor family of ligands was reported to be involved in perineural invasion in pancreatic cancer. Artemin is one member of the glial cell line-derived neurotrophic factor family of ligands. Although Artemin has previously been demonstrated to promote invasiveness of pancreatic cancer, the mechanisms remain poorly understood. In this study, we performed an analysis to determine the effects of Artemin on modulating tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer. We indicated that Artemin and CXCR4 were overexpressed in cancer tissues and widely expressed in pancreatic cancer cell lines. We observed that activation of ERK1/2 and Akt in Artemin-treated cells led to enhanced nuclear accumulation of NF-κB, which then induced CXCR4 expression. Through regulation of the expression of CXCR4, Artemin functionally promoted the migration and invasion in pancreatic cancer cells. The present study indicated that Artemin induced CXCR4 expression by activating Akt and ERK 1/2/NF-κB signaling, thereby modulating tumor cell metastatic potential and invasion activity in pancreatic cancer by regulating SDF-1α/CXCR4 axis. Artemin might be an effective and potent therapeutic target for pancreatic cancer metastasis, especially in perineural invasion.

Inflammatory and neuropathic cold allodynia are selectively mediated by the neurotrophic factor receptor GFRα3.

Tissue injury prompts the release of a number of proalgesic molecules that induce acute and chronic pain by sensitizing pain-sensing neurons (nociceptors) to heat and mechanical stimuli. In contrast, many proalgesics have no effect on cold sensitivity or can inhibit cold-sensitive neurons and diminish cooling-mediated pain relief (analgesia). Nonetheless, cold pain (allodynia) is prevalent in many inflammatory and neuropathic pain settings, with little known of the mechanisms promoting pain vs. those dampening analgesia. Here, we show that cold allodynia induced by inflammation, nerve injury, and chemotherapeutics is abolished in mice lacking the neurotrophic factor receptor glial cell line-derived neurotrophic factor family of receptors-α3 (GFRα3). Furthermore, established cold allodynia is blocked in animals treated with neutralizing antibodies against the GFRα3 ligand, artemin. In contrast, heat and mechanical pain are unchanged, and results show that, in striking contrast to the redundant mechanisms sensitizing other modalities after an insult, cold allodynia is mediated exclusively by a single molecular pathway, suggesting that artemin-GFRα3 signaling can be targeted to selectively treat cold pain.

Ter-cell, A New Target for Hepatocellular Carcinoma Therapy.

Hepatocellular carcinoma (HCC), a malignancy of the liver, has become the second most lethal cause of cancer death globally. Recently, scientists discovered that a splenic erythroblast-like cell induced by the primary tumor, termed Ter-cell, promoted HCC progression and metastasis. These findings shed light on the inhibition of Ter-cell or artemin that can serve as a new therapeutic target for HCC.

Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush.

Recovery after a spinal cord injury often requires that axons restore synaptic connectivity with denervated targets several centimeters from the site of injury. Here we report that systemic artemin (ARTN) treatment promotes the regeneration of sensory axons to the brainstem after brachial dorsal root crush in adult rats. ARTN not only stimulates robust regeneration of large, myelinated sensory axons to the brainstem, but also promotes functional reinnervation of the appropriate target region, the cuneate nucleus. ARTN signals primarily through the RET tyrosine kinase, an interaction that requires the nonsignaling coreceptor GDNF family receptor (GFRα3). Previous studies reported limited GFRα3 expression on large sensory neurons, but our findings demonstrate that ARTN promotes robust regeneration of large, myelinated sensory afferents. Using a cell sorting technique, we demonstrate that GFRα3 expression is similar in myelinated and unmyelinated adult sensory neurons, suggesting that ARTN likely induces long-distance regeneration by binding GFRα3 and RET. Although ARTN is delivered for just 2 wk, regeneration to the brainstem requires more than 3 mo, suggesting that brief trophic support may initiate intrinsic growth programs that remain active until targets are reached. Given its ability to promote targeted functional regeneration to the brainstem, ARTN may represent a promising therapy for restoring sensory function after spinal cord injury.

Quantitative analysis of receptor tyrosine kinase-effector coupling at functionally relevant stimulus levels.

A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5-10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome.

Real-time monitoring of artemin in vivo chaperone activity using luciferase as an intracellular reporter.

Artemin is an abundant thermostable protein in Artemia encysted embryos and considered as a stress protein, as its highly regulated expression is associated with stress resistance. Artemin cDNA was previously isolated and cloned from Artemia urmiana and artemin was found as an efficient molecular chaperone in vitro. Here, co-transformation of E. coli was performed with two expression vectors containing artemin and firefly luciferase for in vivo studies. The time-course of luciferase inactivation at low and elevated temperatures showed that luciferase was rapidly inactivated in control cells, but it was found that luciferase was protected significantly in artemin expressing cells. More interestingly, luciferase activity was completely regained in heat treated artemin expressing cells at room temperature. In addition, in both stress conditions, similar to residual activity of luciferase, cell viability in induced cultures over-expressing artemin was significantly higher than non-expressed artemin cells. It can be suggested that artemin confers impressive resistance in stressful conditions when introduced into E. coli cells, which is due to that it protects proteins against aggregation. Such luciferase co-expression system can be used as a real-time reporter to investigate the activity of chaperone proteins in vivo and provide a rapid and simple test for molecular chaperones.

Investigating the Role of Artemin Glycosylation.

PURPOSE: Oligosaccharides play diverse and unpredictable functional roles when attached to proteins and are a largely unexplored scaffold for deconstructing and attributing novel functions to proteins during drug development. Here, the glycoprotein Artemin (ART) was carefully assessed by multiple analytical methods that allow us to provide a comprehensive understanding of how N-linked glycosylation impact the structural and functional properties of ART. METHODS: Modification of the N-linked glycan of ART was performed by incubation with various enzymes. Biological assays and systems were used to examine the relative activity and pharmacokinetic properties of ART as a function of glycosylation. In order to reveal the conformational impact of glycosylation on ART, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed in addition to differential scanning calorimetry. The colloidal stability of ART glycovariants was assessed by dynamic light scattering, viscometry, and solubility assays. RESULTS: No difference in pharmacokinetics or relative potency was revealed between glycosylated and nonglycosylated ART. Surprisingly, the HDX-MS data indicated that the glycan does not greatly influence the conformation and dynamics of the protein. In contrast, differences in thermal and colloidal stability clearly revealed a role of glycosylation in increasing the solubility and stability of ART. CONCLUSIONS: Our findings demonstrate how careful analysis using multiple advanced techniques can be used to identify and dissect the multiple potential functions of protein glycosylation and form a prerequisite for glycoengineering and drug development of glycoproteins.

[Glial cell line-derived neurotrophic factor family ligands and their therapeutic potential].

Four glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) have been characterized: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These proteins support and restore multiple neuronal populations such as dopaminergic, sensory, motor, hippocampal, basal forebrain, enteric, sympathetic and parasympathetic neurons. Therefore, GFLs attracted significant attention as a potential cure for the diseases caused by neuronal injury and degeneration. Results of multiple experiments indicate that GFLs can alleviate behavioral symptoms and restore affected neurons in animal models of several neurological disorders including, among others, Parkinson's disease (PD). During the last decade, GDNF protein and NRTN gene therapy have been tested in several clinical trials in patients with PD. Although the results of phase I clinical trials were positive, phase II clinical trials failed to reach primary end-points. Poor pharmacokinetic properties of GFLs (inability to penetrate tissues barriers, high affinity for extracellular matrix, etc.) could contribute to the absence of clear clinical benefits of these proteins for the patients. The purpose of this paper was to review therapeutic potential of GFLs and discuss possibilities to overcome difficulties associated with pharmacokinetic properties and delivery of GFLs to target neurons.