Esterified Cholesterol Is Highly Localized to Bruch's Membrane, as Revealed by Lipid Histochemistry in Wholemounts of Human Choroid

Accumulation of neutral lipids in Bruch''s membrane (BrM) is a major age change in human retina and contributes to the formation of extracellular lesions associated with age-related macular degeneration. We developed a BrM–choroid wholemounting technique suitable for reliable staining and evaluated different fluorescent lipid dyes for topographic semiquantitative analysis of BrM lipids. Thin BrM–choroid complexes with partially stripped choroid from 10 aged donor eyes were prepared with an optimized wholemounting technique. Preparation quality was monitored by examining 1-μm-thick sections of representative samples. The staining patterns of Nile Red, BODIPY 493/503, filipin for unesterified cholesterol (UC-F), filipin for esterified cholesterol (EC-F), and Oil Red O in wholemounts were compared with their staining patterns in chorioretinal sections, using wide-field epi-fluorescence microscopy. Wholemounts exhibited optimal flatness on the BrM side. Reduced tissue thickness allowed reliable dye penetration and staining of BrM. Only EC-F was with high specificity localized to BrM and demonstrated an intense and distinct granular staining pattern not previously appreciated in chorioretinal sections. All other lipid dyes also stained choroidal or retinal tissue intensely. No dye provided perfect characteristics in regard to representing all neutral lipid classes present in BrM or to fluorescence intensity. Nevertheless, only EC-F was highly localized to BrM with a specific granular pattern. Because direct assays indicate that esterified cholesterol is abundantly present in BrM, we consider EC-F the most valuable choice for analyzing neutral lipid deposits in human BrM. (J Histochem Cytochem 57:731–739, 2009)     

Apolipoprotein B-containing lipoproteins in retinal aging and age-related macular degeneration

The largest risk factor for age-related macular degeneration (ARMD) is advanced age. With aging, there is a striking accumulation of neutral lipids in Bruch''s membrane (BrM) of normal eye that continues through adulthood. This accumulation has the potential to significantly impact the physiology of the retinal pigment epithelium (RPE). It also ultimately leads to the creation of a lipid wall at the same locations where drusen and basal linear deposit, the pathognomonic extracellular, lipid-containing lesions of ARMD, subsequently form. Here, we summarize evidence obtained from light microscopy, ultrastructural studies, lipid histochemistry, assay of isolated lipoproteins, and gene expression analysis. These studies suggest that lipid deposition in BrM is at least partially due to accumulation of esterified cholesterol-rich, apolipoprotein B-containing lipoprotein particles produced by the RPE. Furthermore, we suggest that the formation of ARMD lesions and their aftermath may be a pathological response to the retention of a sub-endothelial apolipoprotein B lipoprotein, similar to a widely accepted model of atherosclerotic coronary artery disease (Tabas, I., K. J. Williams, and J. Borén. 2007. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation. 116:1832–1844). This view provides a conceptual basis for the development of novel treatments that may benefit ARMD patients in the future.     

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet’s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet''s membrane) is introduced onto the recipient''s posterior stroma³. Unlike Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity⁴. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision¹. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet''s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK⁵. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.     

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (α-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, α-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated α-syn is a neuropathological feature of Parkinson''s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble α-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of α-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated α-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal α-syn biology in transgenic animal models harbouring different α-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.     

Assaying ��-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the β-amyloid peptide (Aβ) is generally believed to be central to the induction of Alzheimer''s disease, but the relevant mechanism(s) of toxicity are still unclear. Aβ is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human Aβ. Depending on the tissue or timing of Aβ expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of Aβ toxicity. For example, transgenic worms with pan-neuronal Aβ expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an Aβ transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter Aβ toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.     

Isolation,Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.     

3D iPSC modeling of the retinal pigment epithelium-choriocapillaris complex identifies factors involved in the pathology of macular degeneration

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Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain¹. The discovery in 2004 of mutations in LRRK2 that cause Parkinson''s disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state^2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities^4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations^7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool^9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson''s is open to debate.A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads^4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK2¹². Recently, Daniëls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available¹³. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including α-synuclein, Moesin and 4-EBP^14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.     

Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective degeneration of dopaminergic neurons in the nigrostriatal pathway. Several lines of evidence indicate that mitochondrial dysfunction contributes to its etiology. Other studies have suggested that alterations in sterol homeostasis correlate with increased risk for PD. Whether these observations are functionally related is, however, unknown. In this study, we used a toxin-induced mouse model of PD and measured levels of nine sterol intermediates. We found that lanosterol is significantly (～50%) and specifically reduced in the nigrostriatal regions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, indicative of altered lanosterol metabolism during PD pathogenesis. Remarkably, exogenous addition of lanosterol rescued dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in culture. Furthermore, we observed a marked redistribution of lanosterol synthase from the endoplasmic reticulum to mitochondria in dopaminergic neurons exposed to MPP+, suggesting that lanosterol might exert its survival effect by regulating mitochondrial function. Consistent with this model, we find that lanosterol induces mild depolarization of mitochondria and promotes autophagy. Collectively, our results highlight a novel sterol-based neuroprotective mechanism with direct relevance to PD.     

Imaging multiple phases of neurodegeneration: a novel approach to assessing cell death in vivo

Nerve cell death is the key event in all neurodegenerative disorders, with apoptosis and necrosis being central to both acute and chronic degenerative processes. However, until now, it has not been possible to study these dynamically and in real time. In this study, we use spectrally distinct, well-recognised fluorescent cell death markers to enable the temporal resolution and quantification of the early and late phases of apoptosis and necrosis of single nerve cells in different disease models. The tracking of single-cell death profiles in the same living eye over hours, days, weeks and months is a significant advancement on currently available techniques. We identified a numerical preponderance of late-phase versus early-phase apoptotic cells in chronic models, reinforcing the commonalities between cellular mechanisms in different disease models. We showed that MK801 effectively inhibited both apoptosis and necrosis, but our findings support the use of our technique to investigate more specific anti-apoptotic and anti-necrotic strategies with well-defined targets, with potentially greater clinical application. The optical properties of the eye provide compelling opportunities for the quantitative monitoring of disease mechanisms and dynamics in experimental neurodegeneration. Our findings also help to directly observe retinal nerve cell death in patients as an adjunct to refining diagnosis, tracking disease status and assessing therapeutic intervention.     

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.     

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or “scarring” is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first “organ” culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.     

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson''s disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 ¹. Because ageing is the most important risk factor, cases of PD will increase during the next decades ². Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD ^3-11.After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.     

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease¹. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms² and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson''s disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.     

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples² (Fig.1). A 12 μm thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity². Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson''s disease (PD). PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age^3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA⁵. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)^1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA⁷. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control^7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson''s disease and L-DOPA induced dyskinesia. MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin''s opioid-receptor binding capacity⁹. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.