Vertigoheel promotes rodent cognitive performance in multiple memory tests.

Introduction: Cognitive impairment associated with old age or various brain disorders may be very disabling for affected individuals, placing their carers and public health services under considerable stress. The standard-of-care drugs produce only transient improvement of cognitive impairment in older people, so the search for novel, safe and effective therapeutics that would help to reverse or delay cognitive impairment is warranted. Repurposing pharmacological therapies with well-established safety record for additional indications is a promising recent trend in drug development. Vertigoheel (VH-04), a multicomponent drug made of Ambra grisea, Anamirta cocculus L., Conium maculatum, and Petroleum rectificatum, has been successfully used for several decades in the treatment of vertigo. Here, we investigated effects of VH-04 on cognitive performance in standard behavioral tests assessing different types of memory and explored cellular and molecular underpinnings of VH-04's biological activity. Methods: In the majority of behavioral experiments, namely in the spontaneous and rewarded alternation tests, passive avoidance test, contextual/cued fear conditioning, and social transmission of food preference, we examined the ability of single and repeated intraperitoneal administrations of VH-04 to improve cognitive parameters of mice and rats disrupted by the application of the muscarinic antagonist scopolamine. In addition, we also assessed how VH-04 affected novel object recognition and influenced performance of aged animals in Morris water maze. Furthermore, we also studied the effects of VH-04 on primary hippocampal neurons in vitro and mRNA expression of synaptophysin in the hippocampus. Results: Administration of VH-04 positively influenced visual recognition memory in the novel object recognition test and alleviated the impairments in spatial working memory and olfactory memory caused by the muscarinic antagonist scopolamine in the spontaneous alternation and social transmission of food preference tests. In addition, VH-04 improved retention of the spatial orientation memory of old rats in the Morris water maze. In contrast, VH-04 did not have significant effects on scopolamine-induced impairments in tests of fear-aggravated memory or rewarded alternation. Experiments in vitro showed that VH-04 stimulated neurite growth and possibly reversed the age-dependent decrease in hippocampal synaptophysin mRNA expression, which implies that VH-04 may preserve synaptic integrity in the aging brain. Discussion: Our findings allow a cautious conclusion that in addition to its ability to alleviate manifestations of vertigo, VH-04 may be also used as a cognitive enhancer.

NADH Fluorescence Lifetime Imaging Microscopy Reveals Selective Mitochondrial Dysfunction in Neurons Overexpressing Alzheimer's Disease-Related Proteins.

Alzheimer's disease (AD), the most prevalent form of dementia, affects globally more than 30 million people suffering from cognitive deficits and neuropsychiatric symptoms. Substantial evidence for the involvement of mitochondrial dysfunction in the development and/or progression of AD has been shown in addition to the pathological hallmarks amyloid beta (Aß) and tau. Still, the selective vulnerability and associated selective mitochondrial dysfunction cannot even be resolved to date. We aimed at optically quantifying mitochondrial function on a single-cell level in primary hippocampal neuron models of AD, unraveling differential involvement of cell and mitochondrial populations in amyloid precursor protein (APP)-associated mitochondrial dysfunction. NADH lifetime imaging is a highly sensitive marker-free method with high spatial resolution. However, deciphering cellular bioenergetics of complex cells like primary neurons has still not succeeded yet. To achieve this, we combined highly sensitive NADH lifetime imaging with respiratory inhibitor treatment, allowing characterization of mitochondrial function down to even the subcellular level in primary neurons. Measuring NADH lifetime of the same neuron before and after respiratory treatment reveals the metabolic delta, which can be taken as a surrogate for cellular redox capacity. Correlating NADH lifetime delta with overexpression strength of Aß-related proteins on the single-cell level, we could verify the important role of intracellular Aß-mediated mitochondrial toxicity. Subcellularly, we could demonstrate a higher respiration in neuronal somata in general than dendrites, but a similar impairment of somatic and dendritic mitochondria in our AD models. This illustrates the power of NADH lifetime imaging in revealing mitochondrial function on a single and even subcellular level and its potential to shed light into bioenergetic alterations in neuropsychiatric diseases and beyond.

Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH, FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques.

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.

Ubiquitination and the proteasome rather than caspase-3-mediated C-terminal cleavage are involved in the EAAT2 degradation by staurosporine-induced cellular stress.

Diminished glutamate (Glu) uptake via the excitatory amino acid transporter EAAT2, which normally accounts for ~90% of total forebrain EAAT activity, may contribute to neurodegeneration via Glu-mediated excitotoxicity. C-terminal cleavage by caspase-3 (C3) was reported to mediate EAAT2 inactivation and down-regulation in the context of neurodegeneration. For a detailed analysis of C3-dependent EAAT2 degradation, we employed A172 glioblastoma as well as hippocampal HT22 cells and murine astrocytes over-expressing VSV-G-tagged EAAT2 constructs. C3 activation was induced by staurosporine (STR). In HT22 cells, STR-induced C3 activation-induced rapid EAAT2 protein degradation. The mutation of asparagine 504 to aspartate (D504N), which should inactivate the putative C3 cleavage site, increased EAAT2 activity in A172 cells. In contrast, the D504N mutation did not protect EAAT2 protein against STR-induced degradation in HT22 cells, whereas inhibition of caspases, ubiquitination and the proteasome did. Similar results were obtained in astrocytes. Phylogenetic analysis showed that C-terminal ubiquitin acceptor sites-but not the putative C3 cleavage site-exhibit a high degree of conservation. Moreover, C-terminal truncation mimicking C3 cleavage increased rather than decreased EAAT2 activity and stability as well as protected EAAT2 against STR-induced ubiquitination-dependent degradation. We conclude that cellular stress associated with endogenous C3 activation degrades EAAT2 via a pathway involving ubiquitination and the proteasome but not direct C3-mediated cleavage. In addition, C3 cleavage of EAAT2, described to occur in other models, is unlikely to inactivate EAAT2. However, mutation of the highly conserved D504 within the putative C3 cleavage site increases EAAT2 activity via an unknown mechanism.

In Vivo Protein Complementation Demonstrates Presynaptic α-Synuclein Oligomerization and Age-Dependent Accumulation of 8-16-mer Oligomer Species.

Intracellular accumulation of α-synuclein (α-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson's disease (PD) and related α-synucleinopathies. Oligomerization and spreading of α-syn from neuron to neuron have been suggested as key events contributing to the progression of PD. To directly visualize and characterize α-syn oligomerization and spreading in vivo, we generated two independent conditional transgenic mouse models based on α-syn protein complementation assays using neuron-specifically expressed split Gaussia luciferase or split Venus yellow fluorescent protein (YFP). These transgenic mice allow direct assessment of the quantity and subcellular distribution of α-syn oligomers in vivo. Using these mouse models, we demonstrate an age-dependent accumulation of a specific subtype of α-syn oligomers. We provide in vivo evidence that, although α-syn is found throughout neurons, α-syn oligomerization takes place at the presynapse. Furthermore, our mouse models provide strong evidence for a transsynaptic cell-to-cell transfer of de novo generated α-syn oligomers in vivo.

The role of PTB domain containing adaptor proteins on PICALM-mediated APP endocytosis and localization.

One hallmark of Alzheimer's disease (AD) is the presence of amyloid plaques, which mainly consist of the amyloid precursor protein (APP) cleavage product amyloid ß (Aß). For cleavage to occur, the APP must be endocytosed from the cell surface. The phosphatidylinositol binding clathrin assembly protein (PICALM) is involved in clathrin-mediated endocytosis and polymorphisms in and near the gene locus were identified as genetic risk factors for AD. PICALM overexpression enhances APP internalization and Aß production. Furthermore, PICALM shuttles into the nucleus, but its function within the nucleus is still unknown. Using co-immunoprecipitation, we demonstrated an interaction between PICALM and APP, which is abrogated by mutation of the APP NPXY-motif. Since the NPXY-motif is an internalization signal that binds to phosphotryrosine-binding domain-containing adaptor proteins (PTB-APs), we hypothesized that PTB-APs can modulate the APP-PICALM interaction. We found that interaction between PICALM and the PTB-APs (Numb, JIP1b and GULP1) enhances the APP-PICALM interaction. Fluorescence activated cell sorting analysis and internalization assays revealed differentially altered APP cell surface levels and endocytosis rates that depended upon the presence of PICALM and co-expression of distinct PTB-APs. Additionally, we were able to show an impact of PICALM nuclear shuttling upon co-expression of PTB-APs and PICALM, with the magnitude of the effect depending on which PTB-AP was co-expressed. Taken together, our results indicate a modulating effect of PTB-APs on PICALM-mediated APP endocytosis and localization.

NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research.

More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker-free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research. © 2018 International Society for Advancement of Cytometry.

sAPPß and sAPPα increase structural complexity and E/I input ratio in primary hippocampal neurons and alter Ca2+ homeostasis and CREB1-signaling.

One major pathophysiological hallmark of Alzheimer's disease (AD) is senile plaques composed of amyloid ß (Aß). In the amyloidogenic pathway, cleavage of the amyloid precursor protein (APP) is shifted towards Aß production and soluble APPß (sAPPß) levels. Aß is known to impair synaptic function; however, much less is known about the physiological functions of sAPPß. The neurotrophic properties of sAPPα, derived from the non-amyloidogenic pathway of APP cleavage, are well-established, whereas only a few, conflicting studies on sAPPß exist. The intracellular pathways of sAPPß are largely unknown. Since sAPPß is generated alongside Aß by ß-secretase (BACE1) cleavage, we tested the hypothesis that sAPPß effects differ from sAPPα effects as a neurotrophic factor. We therefore performed a head-to-head comparison of both mammalian recombinant peptides in developing primary hippocampal neurons (PHN). We found that sAPPα significantly increases axon length (p = 0.0002) and that both sAPPα and sAPPß increase neurite number (p < 0.0001) of PHN at 7 days in culture (DIV7) but not at DIV4. Moreover, both sAPPα- and sAPPß-treated neurons showed a higher neuritic complexity in Sholl analysis. The number of glutamatergic synapses (p < 0.0001), as well as layer thickness of postsynaptic densities (PSDs), were significantly increased, and GABAergic synapses decreased upon sAPP overexpression in PHN. Furthermore, we showed that sAPPα enhances ERK and CREB1 phosphorylation upon glutamate stimulation at DIV7, but not DIV4 or DIV14. These neurotrophic effects are further associated with increased glutamate sensitivity and CREB1-signaling. Finally, we found that sAPPα levels are significantly reduced in brain homogenates of AD patients compared to control subjects. Taken together, our data indicate critical stage-dependent roles of sAPPs in the developing glutamatergic system in vitro, which might help to understand deleterious consequences of altered APP shedding in AD patients, beyond Aß pathophysiology.

Mitochondrial matrix pH as a decisive factor in neurometabolic imaging.

Alterations of cellular bioenergetics are a common feature in most neurodegenerative disorders. However, there is a selective vulnerability of different brain regions, cell types, and even mitochondrial populations to these metabolic disturbances. Thus, the aim of our study was to establish and validate an in vivo metabolic imaging technique to screen for mitochondrial function on the subcellular level. Based on nicotinamide adenine dinucleotide (phosphate) fluorescence lifetime imaging microscopy [NAD(P)H FLIM], we performed a quantitative correlation to high-resolution respirometry. Thereby, we revealed mitochondrial matrix pH as a decisive factor in imaging NAD(P)H redox state. By combining both parameters, we illustrate a quantitative, high-resolution assessment of mitochondrial function in metabolically modified cells as well as in an amyloid precursor protein-overexpressing model of Alzheimer's disease. Our metabolic imaging technique provides the basis for dissecting mitochondrial deficits not only in a range of neurodegenerative diseases, shedding light onto bioenergetic failures of cells remaining in their metabolic microenvironment.

The Golgi-localized, gamma ear-containing, ARF-binding (GGA) protein family alters alpha synuclein (α-syn) oligomerization and secretion.

Several age-related neurodegenerative disorders are associated with protein misfolding and aggregation of toxic peptides. α-synuclein (α-syn) aggregation and the resulting cytotoxicity is a hallmark of Parkinson's disease (PD) as well as dementia with Lewy bodies. Rising evidence points to oligomeric and pre-fibrillar forms as the pathogenic species, and oligomer secretion seems to be crucial for the spreading and progression of PD pathology. Recent studies implicate that dysfunctions in endolysosomal/autophagosomal pathways increase α-syn secretion. Mutation in the retromer-complex protein VPS35, which is involved in endosome to Golgi transport, was suggested to cause familial PD. GGA proteins regulate vesicular traffic between Golgi and endosomes and might work as antagonists for retromer complex mediated transport. To investigate the role of the GGAs in the α-syn oligomerization and/or secretion process we utilized protein-fragment complementation assays (PCA). We here demonstrate that GGAs alter α-syn oligomer secretion and α-syn oligomer-mediated toxicity. Specifically, we determined that GGA3 modifies extracellular α-syn species in an exosome-independent manner. Our data suggest that GGA3 drives α-syn oligomerization in endosomal compartments and thus facilitates α-syn oligomer secretion. Preventing the early events in α-syn oligomer release may be a novel approach to halt disease spreading in PD and other synucleinopathies.

Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration.

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aß), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aß and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aß, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aß in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aß levels or Aß alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aß levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aß-mediated mitochondrial toxicity. Analyzing Aß localization revealed that intracellular levels of Aß and an increased spatial association of APP/Aß with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aß accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases.

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-ß Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1).

Proteolytic processing of amyloid-ß precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aß), which accumulates in senile plaques in Alzheimer's disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aß generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPß, and Aß upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

Engulfment adapter PTB domain containing 1 interacts with and affects processing of the amyloid-ß precursor protein.

Previous studies identified engulfment adapter phosphotyrosine binding (PTB) domain containing 1 (GULP1) as an NPXY-motif interactor of low-density lipoprotein receptor-related protein 1 (LRP1) and suggested a potential relevance in Alzheimer's disease (AD). Since AD associated proteins amyloid-ß A4 precursor protein (APP) and LRP1 were shown to interact with the PTB domain of Fe65 and several other adapters via their intracellular NPXY-motifs, we examined a possible interaction of GULP1 PTB domain with the YENPTY-motif of APP. Here we demonstrate that GULP1 is present in human hippocampal and neocortical neurons. Confocal live cell imaging revealed that coexpressed and endogenous GULP1 colocalizes with APP in the Golgi and endoplasmic reticulum. Analysis of the interacting domains by co-immunoprecipitation of point and deletion mutants revealed that the interaction depends on the PTB domain of GULP1 and the YENPTY-motif of APP. Coexpression of GULP1 affected APP cell surface localization and suppressed generation of Aß40/42 and sAPPα. Taken together, these data identify GULP1 as a novel neuronal APP interacting protein that alters trafficking and processing of APP.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between ß-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1.

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Abeta), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for beta-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPbeta, but not sAPPalpha. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that--in addition to its role in APP trafficking--LRP1 affects APP processing by competing for cleavage by BACE1.

Impact of cholesterol level upon APP and BACE proximity and APP cleavage.

Cleavage of APP by BACE is the first proteolytic step in the production of Amyloid beta (Abeta, which accumulates in senile plaques in Alzheimer's disease. BACE-cleavage of APP is thought to happen in endosomes. However, there are controversial data whether APP and BACE can already interact on the cell surface dependent on the cholesterol level. To examine whether APP and BACE come into close proximity on the cell surface in living cells, we employed a novel technique by combining time-resolved Förster resonance energy transfer (FRET) measurements with total internal reflection microscopy (TIRET microscopy). Our data indicate that BACE and APP come into close proximity within the cell, but probably not on the cell surface. To analyze the impact of alterations in cholesterol level upon BACE-cleavage, we measured sAPP secretion. Alteration of APP processing and BACE proximity by cholesterol might be explained by alterations in cell membrane fluidity.