Ginkgo biloba leaf extract EGb 761® as a paragon of the product by process concept.

It is an often-neglected fact that extracts derived from the very same plant can differ significantly in their phytochemical composition, and thus also in their pharmacokinetic and pharmacodynamic properties which are the basis for their clinical efficacy and safety. The Ginkgo biloba L. [Ginkgoaceae] special extract EGb 761® is one of the best-studied plant extracts in the world. In the present review, using that extract as a paradigm, we describe insights how climate, the harvest region, processing of the plant material, the drying process, the extraction solvents, and the details of the subsequent process steps substantially impact the quality and uniformity of the final extract. We highlight the importance of regulating active constituent levels and consistent reduction of undesired substances in herbal extracts. This is accomplished by a controlled production process and corresponding analytical specifications. In conclusion, since extracts derived from the same plant can have very different phytochemical compositions, results from pharmacological, toxicological and clinical studies gained with one specific extract cannot be extrapolated to other extracts that were generated using different production processes. We propose that the heterogenous nature of extracts should be meticulously considered when evaluating the efficacy and safety of plant-derived remedies.

Alleviation of Post-COVID-19 Cognitive Deficits by Treatment with EGb 761®: A Case Series.

BACKGROUND Cognitive symptoms persisting longer than 3 months after infection, such as memory loss, or difficulties concentrating, have been reported in up to one-third of patients after COVID-19. Evidence-based therapeutic interventions to treat post-COVID-19 symptoms (also called "Long-COVID symptoms") have not yet been established, and the treating physicians must rely on conjecture to help patients. Based on its mechanism of action and its efficacy in treating cognitive impairment, as well as its good tolerability, the Ginkgo biloba special extract EGb 761 has been suggested as a remedy to alleviate cognitive post-COVID-19 symptoms. In many studies, EGb 761 has been demonstrated to protect endothelial cells, to have potent anti-inflammatory effects, and to enhance neuroplasticity. CASE REPORT Here, we report for the first time the application of EGb 761 in the therapy of post-COVID-19-related cognitive deficits. Three women and 2 men, aged 26 to 59 years (average age 34.6 years), presented with concentration and attention deficits, cognitive deficiencies, and/or fatigue 9-35 weeks after infection. A daily dose of 2×80 mg of EGb 761 did not cause any detectable adverse effects, and it substantially improved or completely restored cognitive deficits and, when initially present, also other symptoms, such as fatigue and hyposmia, within an observation period of up to 6 months. CONCLUSIONS Our observations support the hypothesis that EGb 761 might be a low-risk treatment option for post-COVID-19 patients with cognitive symptoms. Moreover, we derive recommendations for randomized controlled clinical trials to confirm efficacy in that indication.

Pharmacologic treatments in preclinical tinnitus models with special focus on Ginkgo biloba leaf extract EGb 761®.

Tinnitus is defined as the perception of sound in the absence of external acoustic stimuli. Frequent comorbidities or associated factors are depression, anxiety, concentration problems, insomnia, resignation, helplessness, headache, bruxism, or social isolation, just to name a few. Although many therapeutic approaches have already been tested with varying success, there still is no cure available for tinnitus. The search for an effective treatment has been hampered by the fact that the mechanisms of tinnitus development are still not fully understood, although several models are available and discussed in this review. Our review will give a brief overview about preclinical models, presenting the heterogeneity of tinnitus sub-types depending on the different inner ear and brain structures involved in tinnitus etiology and pathogenesis. Based on these models we introduce the different target structures and transmitter systems implicated in tinnitus development and provide an extensive overview on preclinical drug-based therapeutic approaches that have been explored in various animal models. As the special extract from Ginkgo biloba leaves EGb 761® has been the most widely tested drug in both non-clinical tinnitus models as well as in clinical trials, a special focus will be given to EGb 761®. The efficacy of terpene lactones, flavone glycosides and proanthocyanidines with their distinct contribution to the overall efficacy profile of the multi-constituent drug EGb 761® will be discussed.

Moderately aged OFA rats as a novel model for mild age-related alterations in learning and memory.

Aged rodents have been used as preclinical models of age-associated cognitive decline. Most of those models displayed substantial impairments in learning and memory. The initial, more subtle changes that precede more severe losses in cognitive abilities have not been well characterized. Here, we established a model detecting initial subtle cognitive changes by comparing the performance of moderately aged Oncins France Strain A Sprague Dawley rats with young rats in the Morris water maze (MWM) and the Open Field (OF) test. Both age groups improved their performance during the training period at a similar rate; however, the older rats performed worse in several parameters measured in the MWM. Our results suggest that already at the age of 18-20 months rats show changes in their approach to solve the spatial memory task while their ability to learn is not yet diminished. The disparate spatial information processing of the moderately aged rats provides a novel animal model for early age-related cognitive alterations that could be useful to test the effect of early intervention strategies. Moreover, our results suggest that the sensitivity of cognitive tests in the elderly could be substantially enhanced if they assess both the improvement after several trials, and the strategy used to solve a certain task.

Novel Cell-Based Assay for Identification of LRRK2 Inhibitors Using Its Aberrant Regulation of a Pluripotency Gene.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2), such as the G2019S mutation, are the most common cause of familial Parkinson's disease (PD). The G2019S mutation impairs neurite outgrowth. We hypothesized that those effects could be related to an altered expression of pluripotency genes, which may provide a readout for a screening assay based on LRRK2 function. Here, we show that the G2019S mutation mediates a sustained aberrant upregulation of the transcription factors Nanog and Oct4 that in wild-type are downregulated after differentiation. The aberrant regulation of Nanog can be concentration dependently reversed by LRRK2 tool inhibitors. Building on this knowledge, we developed an assay for the identification and assessment of compounds that inhibit the aberrant pathophysiological activity of mutant LRRK2. Furthermore, the aberrant neural pluripotency is consistent with Parkinson's pathophysiology and with the epidemiological association between the G2019S genotype and cancer risk.

Differences in Stress-Induced Modulation of the Auditory System Between Wistar and Lewis Rats.

Many aspects of stress-induced physiological and psychological effects have been characterized in people and animals. However, stress effects on the auditory system are less explored and their mechanisms are not well-understood, in spite of its relevance for a variety of diseases, including tinnitus. To expedite further research of stress-induced changes in the auditory system, here we compare the reactions to stress among Wistar and Lewis rats. The animals were stressed for 24 h, and subsequently we tested the functionality of the outer hair cells (OHCs) using distortion product otoacoustic emissions (DPOAEs) and auditory neurons using evoked auditory brainstem responses (ABR). Lastly, using Western blot, we analyzed the levels of plasticity-related proteins in the inferior colliculus, confirming that the inferior colliculus is involved in the adaptive changes that occur in the auditory system upon stress exposure. Surprisingly, the two strains reacted to stress quite differently: Lewis rats displayed a lowering of their auditory threshold, whereas it was increased in Wistar rats. These functional differences were seen in OHCs of the apical region (low frequencies) and in the auditory neurons (across several frequencies) from day 1 until 2 weeks after the experimental stress ended. Wistar and Lewis rats may thus provide models for auditory threshold increase and decrease, respectively, which can both be observed in different patients in response to stress.

Anti-neuroinflammatory effects of Ginkgo biloba extract EGb761 in LPS-activated primary microglial cells.

BACKGROUND: Neuroinflammation is a key factor of Alzheimer's disease (AD) and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system (CNS). They play an essential role in the maintenance of homeostasis and responses to neuroinflammation. Ginkgo biloba extract EGb 761 is one of the most commonly used natural medicines owing to its established efficacy and remarkable biological activities especially in respect to CNS diseases. However, only few studies have addressed the effects and mechanisms of Ginkgo biloba extract in microglia activation. METHODS: We measured the production of pro-inflammatory mediators and cytokines by ELISA and analyzed gene expressions by qRT-PCR and Western Blot in LPS treated cultured primary rat microglia. RESULTS: The Ginkgo biloba extract EGb 761 significantly inhibited the release of prostaglandin E2 (PGE2) and differentially regulated the levels of pro-inflammatory cytokines. The inhibition of LPS-induced PGE2 release in primary microglia was partially dependent on reduced protein synthesis of mPGES-1 and the reduction in the activation of cytosolic phospholipase A2 (cPLA2) without altering COX-2 enzymatic activity, inhibitor of kappa B alpha (IkappaBalpha) degradation, and the activation of multiple mitogen activated protein kinases (MAPKs). Altogether, we showed that EGb 761 reduces neuro-inflammatory activation in primary microglial cells by targeting PGE2 release and cytokines. CONCLUSION: Ginkgo biloba extract EGb 761 displayed anti-neuroinflammatory activity in LPS-activated primary microglia cells. EGb 761 was able to reduce neuroinflammatory activation by targeting the COX/PGE2 pathway. This effect might contribute to the established clinical cognitive efficacy in Alzheimer's disease, vascular and mixed dementia.

Development of a Cell-Based Assay to Assess Binding of the proNGF Prodomain to Sortilin.

Sortilin was first identified based on its activity as part of intracellular protein sorting machinery. Recently, it was discovered that sortilin also acts as a cell surface receptor for the propeptide form of nerve growth factor (proNGF), progranulin, and neurotensin. The interaction of sortilin to these neurotrophic ligands is linked to diseases of the nervous system that lead to neurodegeneration and neuropathic pain. Blocking of the interaction of sortilin to these ligands may prevent or slow the progress of these nervous system disorders. In vitro screening assays for blocking compounds or peptides are part of the standard set of tools for drug discovery. However, assays for sortilin biology are not readily available to determine if the selected blocking agent inhibits sortilin activity on the surface of cells. We have developed a sortilin specific cell based assay to identify compounds that specifically block interaction between sortilin and proNGF prodomain. The assay system records both the presence of sortilin on the cell surface and the interaction with the pro domain of NGF. Fluorescent images of the sortilin expressing cells are analyzed for the presence of pro domain of NGF. Sortilin-positive and sortilin-negative cells within one well are concomitantly and automatically analyzed. Sortilin-pro domain interaction can be blocked dose dependently by neurotensin and synthetic compounds. The assay will facilitate the discovery of entities interfering with the binding of sortilin to the NGF pro domain. This assay can be modified to screen for inhibitors of the binding of ligands to other complex cell surface receptors.

Protection of Primary Dopaminergic Midbrain Neurons by GPR139 Agonists Supports Different Mechanisms of MPP(+) and Rotenone Toxicity.

The G-protein coupled receptor 139 (GPR139) is expressed specifically in the brain in areas of relevance for motor control. GPR139 function and signal transduction pathways are elusive, and results in the literature are even contradictory. Here, we examined the potential neuroprotective effect of GPR139 agonism in primary culture models of dopaminergic (DA) neuronal degeneration. We find that in vitro GPR139 agonists protected primary mesencephalic DA neurons against 1-methyl-4-phenylpyridinium (MPP(+))-mediated degeneration. Protection was concentration-dependent and could be blocked by a GPR139 antagonist. However, the protection of DA neurons was not found against rotenone or 6-hydroxydopamine (6-OHDA) mediated degeneration. Our results support differential mechanisms of toxicity for those substances commonly used in Parkinson's disease (PD) models and potential for GPR139 agonists in neuroprotection.

Computer-aided discovery of aromatic l-α-amino acids as agonists of the orphan G protein-coupled receptor GPR139.

GPR139 is an orphan G protein-coupled receptor expressed mainly in the central nervous system. We developed a pharmacophore model based on known GPR139 surrogate agonists which led us to propose aromatic-containing dipeptides as potential ligands. Upon testing, the dipeptides demonstrated agonism in the Gq pathway. Next, in testing all 20 proteinogenic l-α-amino acids, L-tryptophan and l-phenylalanine were found to have EC50 values of 220 and 320 µM, respectively, making them the first putative endogenous agonists for GPR139.

Transduction of neural precursor cells with TAT-heat shock protein 70 chaperone: therapeutic potential against ischemic stroke after intrastriatal and systemic transplantation.

Novel therapeutic concepts against cerebral ischemia focus on cell-based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell-based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein-transduced with TAT-heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT-Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT-Hsp70-transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT-Hsp70-transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood-brain-barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long-term neuroprotection.

Protection by neuroglobin and cell-penetrating peptide-mediated delivery in vivo: a decade of research. Comment on Cai et al: TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice. Exp Neurol. 2011; 227(1): 224-31.

Over the last decade, numerous studies have suggested that neuroglobin is able to protect against the effects of ischemia. However, such results have mostly been based on models using transgenic overexpression or viral delivery. As a therapy, new technology would need to be applied to enable delivery of high concentrations of neuroglobin shortly after the patient suffers the stroke. An approach to deliver proteins in ischemia in vivo in a timely manner is the use of cell-penetrating peptides (CPP). CPP have been used in animal models for brain diseases for about a decade as well. In a recent issue of Experimental Neurology, Cai and colleagues test the effect of CPP-coupled neuroglobin in an in vivo stroke model. They find that the fusion protein protects the brain against the effect of ischemia when applied before stroke onset. Here, a concise review of neuroglobin research and the application of CPP peptides in hypoxia and ischemia is provided.

Transplantation of TAT-Bcl-xL-transduced neural precursor cells: long-term neuroprotection after stroke.

Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x(L) and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x(L)-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x(L)-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x(L) results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.

Cell-penetrating peptide technology to deliver chaperones and associated factors in diseases and basic research.

Cell-penetrating peptides (CPP), also called protein transduction domains (PTD), membrane-permeable peptides (MPP), or Trojan horse peptides, have been used in many different research areas. The delivery of heat shock proteins (Hsp) using CPP has been applied in models for apoptosis, necrosis, oxidative stress, neurodegenerative diseases, stroke, cystic fibrosis, smooth muscle relaxation, myocardial injury, scar formation, and others. This review summarizes the accomplishments of the field over the last years and discusses why Hsp are particularly suitable for CPP-mediated delivery.

Protection of hippocampal neurogenesis by TAT-Bcl-x(L) after cerebral ischemia in mice.

Endogenous neurogenesis persists in the subgranular zone (SGZ) of the adult rodent brain. Cerebral ischemia stimulates endogenous neurogenesis involving proliferation, migration and differentiation of SGZ-derived neural precursor cells (NPC). However, the biological meaning of this phenomenon is limited by poor survival of NPC. In order to study the effects of an acute neuroprotective treatment on hippocampal endogenous neurogenesis after transient cerebral ischemia in mice, we applied a fusion protein consisting of the TAT domain of the HI virus with the anti-apoptotic Bcl-x(L). Intravenous injection of TAT-Bcl-x(L) resulted in reduced hippocampal cell injury for up to 4weeks after stroke as assessed by TUNEL and NeuN staining. This was in line with a TAT-Bcl-x(L)-mediated reduced postischemic microglia activation. Analysis of endogenous hippocampal cell proliferation revealed an increased number of BrdU(+) cells in the TAT-Bcl-x(L) group 4weeks after stroke compared to animals treated with saline and TAT-HA (negative control). Cell proliferation in non-ischemic sham operated animals was not affected by TAT-Bcl-x(L). Twenty-eight days after stroke co-expression of BrdU(+) cells with the immature neuronal marker doublecortin was significantly increased in TAT-Bcl-x(L) animals. Although TAT-Bcl-x(L) treatment also resulted in an increased number of BrdU(+) cells expressing the mature neuronal marker NeuN, the total amount of these cells was low. These data show that TAT-Bcl-x(L) treatment yields both postischemic sustained hippocampal neuroprotection and increased survival of NPC rather than an induction of endogenous neurogenesis itself.

Cell-Penetrating Fragments of the Cdk5 Regulatory Subunit Are Protective in Models of Neurodegeneration.

Cdk5 is essential for neuronal differentiation processes in the brain. Activation of Cdk5 requires the association with the mostly neuron-specific p35 or p39. Overactivation of CDK5 by cleavage of p35 into p25 is thought to be involved in neurodegenerative processes. Here, we have tested an approach to inhibit pathological Cdk5 activation with a Tat-linked dominant-negative fragment of p25. It reduced cell death induced by staurosporine and showed a tendency to alleviate manganese-induced cell death, while it did not protect against 6-OHDA toxicity. Our results suggest that the Tat technique is a suitable tool to inhibit dysregulated CDK5.

Combined inhibition of Cdk5 and ROCK additively increase cell survival, but not the regenerative response in regenerating retinal ganglion cells.

CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.

Tyrosine hydroxylase-positive amacrine interneurons in the mouse retina are resistant against the application of various parkinsonian toxins.

Toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), or rotenone have been used to induce degeneration of dopaminergic (DA) neurons in the nigrostriatal pathway and to reproduce pathological characteristics of Parkinson's disease (PD). DA neurons are also present in the retina, and visual impairments in PD patients have been reported. We examined the vulnerability of TH-positive (TH(+)) amacrine interneurons in the retina against MPTP, 6-OHDA, or rotenone-induced cell death. We intraperitoneally (i.p.) injected mice with MPTP, which induced degeneration of DA neurons in the midbrain. However, no death of TH(+) amacrine cells was detectable in the same mice. HPLC analysis revealed a 9 times lower level of the toxic metabolite of MPTP, MPP(+), in the eye compared with the striatum. Another membrane-permeable compound (Tat-Hsp70) could be delivered into the retina after i.p. application, suggesting that the blood-retina barrier (BRB) could be overcome after systemic application. Possible reason for the survival of retinal amacrine cells after systemic MPTP application was a less efficient conversion into toxic MPP(+) in the retina or a general higher resistance against toxic insults of retinal DA neurons compared with DA neurons in the substantia nigra pars compacta (SNpc). Therefore, we directly injected high doses of MPP(+), 6-OHDA, or rotenone into the eye. No loss of TH(+) amacrine cells in the retina was observed, suggesting different properties and less vulnerability of amacrine neurons compared with DA neurons in the midbrain.

Both systemic and local application of granulocyte-colony stimulating factor (G-CSF) is neuroprotective after retinal ganglion cell axotomy.

BACKGROUND: The hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) plays a crucial role in controlling the number of neutrophil progenitor cells. Its function is mediated via the G-CSF receptor, which was recently found to be expressed also in the central nervous system. In addition, G-CSF provided neuroprotection in models of neuronal cell death. Here we used the retinal ganglion cell (RGC) axotomy model to compare effects of local and systemic application of neuroprotective molecules. RESULTS: We found that the G-CSF receptor is robustly expressed by RGCs in vivo and in vitro. We thus evaluated G-CSF as a neuroprotectant for RGCs and found a dose-dependent neuroprotective effect of G-CSF on axotomized RGCs when given subcutaneously. As stem stell mobilization had previously been discussed as a possible contributor to the neuroprotective effects of G-CSF, we compared the local treatment of RGCs by injection of G-CSF into the vitreous body with systemic delivery by subcutaneous application. Both routes of application reduced retinal ganglion cell death to a comparable extent. Moreover, G-CSF enhanced the survival of immunopurified RGCs in vitro. CONCLUSION: We thus show that G-CSF neuroprotection is at least partially independent of potential systemic effects and provide further evidence that the clinically applicable G-CSF could become a treatment option for both neurodegenerative diseases and glaucoma.