Pharmacoepigenomic Interventions as Novel Potential Treatments for Alzheimer's and Parkinson's Diseases.

Cerebrovascular and neurodegenerative disorders affect one billion people around the world and result from a combination of genomic, epigenomic, metabolic, and environmental factors. Diagnosis at late stages of disease progression, limited knowledge of gene biomarkers and molecular mechanisms of the pathology, and conventional compounds based on symptomatic rather than mechanistic features, determine the lack of success of current treatments, including current FDA-approved conventional drugs. The epigenetic approach opens new avenues for the detection of early presymptomatic pathological events that would allow the implementation of novel strategies in order to stop or delay the pathological process. The reversibility and potential restoring of epigenetic aberrations along with their potential use as targets for pharmacological and dietary interventions sited the use of epidrugs as potential novel candidates for successful treatments of multifactorial disorders involving neurodegeneration. This manuscript includes a description of the most relevant epigenetic mechanisms involved in the most prevalent neurodegenerative disorders worldwide, as well as the main potential epigenetic-based compounds under investigation for treatment of those disorders and their limitations.

Current state of theoretical and experimental studies of the voltage-dependent anion channel (VDAC).

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane provides a controlled pathway for respiratory metabolites in and out of the mitochondria. In spite of the wealth of experimental data from structural, biochemical, and biophysical investigations, the exact mechanisms governing selective ion and metabolite transport, especially the role of titratable charged residues and interactions with soluble cytosolic proteins, remain hotly debated in the field. The computational advances hold a promise to provide a much sought-after solution to many of the scientific disputes around solute and ion transport through VDAC and hence, across the mitochondrial outer membrane. In this review, we examine how Molecular Dynamics, Free Energy, and Brownian Dynamics simulations of the large ß-barrel channel, VDAC, advanced our understanding. We will provide a short overview of non-conventional techniques and also discuss examples of how the modeling excursions into VDAC biophysics prospectively aid experimental efforts. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

MAC inhibitors antagonize the pro-apoptotic effects of tBid and disassemble Bax / Bak oligomers.

Mitochondrial Apoptotic Channel inhibitors or iMACs are di-bromocarbazole derivatives with anti-apoptotic function which have been tested and validated in several mouse models of brain injury and neurodegeneration. Owing to the increased therapeutic potential of these compounds, we sought to expand our knowledge of their mechanism of action. We investigated the kinetics of MAC inhibition in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and semiquantitative western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax and Bak oligomers in the mitochondrial outer membrane. iMACs exert comparable effects on channel conductance of Bax or Bak and similarly affect cytochrome c release from Bax or Bak-containing mitochondria. Interestingly, wild type mitochondria were more susceptible to inhibition than the Bak or Bax knockouts. Western blot analysis showed that wild type mitochondria had lower steady state levels of Bak in the absence of apoptotic stimulation.

Population-based Study of Risk Polymorphisms Associated with Vascular Disorders and Dementia.

INTRODUCTION: Cardiovascular and neurodegenerative disorders are among the major causes of mortality in the developed countries. Population studies evaluate the genetic risk, i.e. the probability of an individual carrying a specific disease-associated polymorphism. Identification of risk polymorphisms is essential for an accurate diagnosis or prognosis of a number of pathologies. AIMS: The aim of this study was to characterize the influence of risk polymorphisms associated with lipid metabolism, hypertension, thrombosis, and dementia, in a large population of Spanish individuals affected by a variety of brain and vascular disorders as well as metabolic syndrome. MATERIAL & METHOD: We performed a cross-sectional study on 4415 individuals from a widespread regional distribution in Spain (48.15% males and 51.85% females), with mental, neurodegenerative, cerebrovascular, and metabolic disorders. We evaluated polymorphisms in 20 genes involved in obesity, vascular and cardiovascular risk, and dementia in our population and compared it with representative Spanish and European populations. Risk polymorphisms in ACE, AGT(235), IL6(573), PSEN1, and APOE (specially the APOE-ε4 allele) are representative of our population as compared to the reference data of Spanish and European individuals. CONCLUSION: The significantly higher distribution of risk polymorphisms in PSEN1 and APOE-ε4 is characteristic of a representative number of patients with Alzheimer's disease; whereas polymorphisms in ACE, AGT(235), and IL6(573), are most probably related with the high number of patients with metabolic syndrome or cerebrovascular damage.

A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer.

Magic angle spinning nuclear magnetic resonance characterization of voltage-dependent anion channel gating in two-dimensional lipid crystalline bilayers.

The N-terminus of the voltage-dependent anion channel (VDAC) has been proposed to contain the mechanistically important gating helices that modulate channel opening and closing. In this study, we utilize magic angle spinning nuclear magnetic resonance (MAS NMR) to determine the location and structure of the N-terminus for functional channels in lipid bilayers by measuring long-range (13)C-(13)C distances between residues in the N-terminus and other domains of VDAC reconstituted into DMPC lipid bilayers. Our structural studies show that the distance between A14 Cß in the N-terminal helix and S193 Cß is ∼4-6 Å. Furthermore, VDAC phosphorylation by a mitochondrial kinase at residue S193 has been claimed to delay mitochondrial cell death by causing a conformational change that closes the channel, and a VDAC-Ser193Glu mutant has been reported to show properties very similar to those of phosphorylated VDAC in a cellular context. We expressed VDAC-S193E and reconstituted it into DMPC lipid bilayers. Two-dimensional (13)C-(13)C correlation experiments showed chemical shift perturbations for residues located in the N-terminus, indicating possible structural perturbations to that region. However, electrophysiological data recorded on VDAC-S193E showed that channel characteristics were identical to those of wild type samples, indicating that phosphorylation of S193 does not directly affect channel gating. The combination of NMR and electrophysiological results allows us to discuss the validity of proposed gating models.

Acidification asymmetrically affects voltage-dependent anion channel implicating the involvement of salt bridges.

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to "gate" between an open and several "closed" states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pKa values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.

Conductance hysteresis in the voltage-dependent anion channel.

Hysteresis in the conductance of voltage-sensitive ion channels is observed when the transmembrane voltage is periodically varied with time. Although this phenomenon has been used in studies of gating of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane for nearly four decades, full hysteresis curves have never been reported, because the focus was solely on the channel opening branches of the hysteresis loops. We studied the hysteretic response of a multichannel VDAC system to a triangular voltage ramp the frequency of which was varied over three orders of magnitude, from 0.5 mHz to 0.2 Hz. We found that in this wide frequency range the area encircled by the hysteresis curves changes by less than a factor of three, suggesting broad distribution of the characteristic times and strongly non-equilibrium behavior. At the same time, quasi-equilibrium two-state behavior is observed for hysteresis branches corresponding to VDAC opening. This enables calculation of the usual equilibrium gating parameters, gating charge and voltage of equipartitioning, which were found to be almost insensitive to the ramp frequency. To rationalize this peculiarity, we hypothesize that during voltage-induced closure and opening the system explores different regions of the complex free energy landscape, and, in the opening branch, follows quasi-equilibrium paths.

Characterization of the Common Genetic Variation in the Spanish Population of Navarre.

Large-scale genomic studies have significantly increased our knowledge of genetic variability across populations. Regional genetic profiling is essential for distinguishing common benign variants from disease-causing ones. To this end, we conducted a comprehensive characterization of exonic variants in the population of Navarre (Spain), utilizing whole genome sequencing data from 358 unrelated individuals of Spanish origin. Our analysis revealed 61,410 biallelic single nucleotide variants (SNV) within the Navarrese cohort, with 35% classified as common (MAF > 1%). By comparing allele frequency data from 1000 Genome Project (excluding the Iberian cohort of Spain, IBS), Genome Aggregation Database, and a Spanish cohort (including IBS individuals and data from Medical Genome Project), we identified 1069 SNVs common in Navarre but rare (MAF ≤ 1%) in all other populations. We further corroborated this observation with a second regional cohort of 239 unrelated exomes, which confirmed 676 of the 1069 SNVs as common in Navarre. In conclusion, this study highlights the importance of population-specific characterization of genetic variation to improve allele frequency filtering in sequencing data analysis to identify disease-causing variants.

Affixing N-terminal α-helix to the wall of the voltage-dependent anion channel does not prevent its voltage gating.

The voltage-dependent anion channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The three-dimensional structure of VDAC1 reveals a channel formed by 19 ß-strands and an N-terminal α-helix located near the midpoint of the pore. The position of this α-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established, but the molecular mechanism continues to be debated; however, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal α-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 that cross-links the α-helix to the wall of the ß-barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal α-helix is located inside the pore of VDAC in the open state and remains associated with ß-strand 11 of the pore wall during voltage gating.

Lipid dynamics and protein-lipid interactions in 2D crystals formed with the ß-barrel integral membrane protein VDAC1.

We employ a combination of (13)C/(15)N magic angle spinning (MAS) NMR and (2)H NMR to study the structural and functional consequences of different membrane environments on VDAC1 and, conversely, the effect of VDAC1 on the structure of the lipid bilayer. MAS spectra reveal a well-structured VDAC1 in 2D crystals of dimyristoylphosphatidylcholine (DMPC) and diphytanoylphosphatidylcholine (DPhPC), and their temperature dependence suggests that the VDAC structure does not change conformation above and below the lipid phase transition temperature. The same data show that the N-terminus remains structured at both low and high temperatures. Importantly, functional studies based on electrophysiological measurements on these same samples show fully functional channels, even without the presence of Triton X-100 that has been found necessary for in vitro-refolded channels. (2)H solid-state NMR and differential scanning calorimetry were used to investigate the dynamics and phase behavior of the lipids within the VDAC1 2D crystals. (2)H NMR spectra indicate that the presence of protein in DMPC results in a broad lipid phase transition that is shifted from 19 to ~27 °C and show the existence of different lipid populations, consistent with the presence of both annular and bulk lipids in the functionally and structurally homogeneous samples.

MAC and Bcl-2 family proteins conspire in a deadly plot.

Apoptosis is an elemental form of programmed cell death; it is fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Apoptosis is also involved in many pathologies including cancer, neurodegenerative diseases, aging, and infarcts. This cell death program is tightly regulated by Bcl-2 family proteins by controlling the formation of the mitochondrial apoptosis-induced channel or MAC. Assembly of MAC corresponds to permeabilization of the mitochondrial outer membrane, which is the so called commitment step of apoptosis. MAC provides the pathway through the mitochondrial outer membrane for the release of cytochrome c and other pro-apoptotic factors from the intermembrane space. While overexpression of anti-apoptotic Bcl-2 eliminates MAC activity, oligomers of the pro-apoptotic members Bax and/or Bak are essential structural component(s) of MAC. Assembly of MAC from Bax or Bak was monitored in real time by directly patch-clamping mitochondria with micropipettes containing the sentinel tBid, a direct activator of Bax and Bak. Herein, a variety of high affinity inhibitors of MAC (iMAC) that may prove to be crucial tools in mechanistic studies have recently been identified. This review focuses on characterization of MAC activity, its regulation by Bcl-2 family proteins, and a discussion of how MAC can be pharmacologically turned on or off depending on the pathology to be treated.

Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.

A lower affinity to cytosolic proteins reveals VDAC3 isoform-specific role in mitochondrial biology.

Voltage-dependent anion channel (VDAC) is the major pathway for the transport of ions and metabolites across the mitochondrial outer membrane. Among the three known mammalian VDAC isoforms, VDAC3 is the least characterized, but unique functional roles have been proposed in cellular and animal models. Yet, a high-sequence similarity between VDAC1 and VDAC3 is indicative of a similar pore-forming structure. Here, we conclusively show that VDAC3 forms stable, highly conductive voltage-gated channels that, much like VDAC1, are weakly anion selective and facilitate metabolite exchange, but exhibit unique properties when interacting with the cytosolic proteins α-synuclein and tubulin. These two proteins are known to be potent regulators of VDAC1 and induce similar characteristic blockages (on the millisecond time scale) of VDAC3, but with 10- to 100-fold reduced on-rates and altered α-synuclein blocking times, indicative of an isoform-specific function. Through cysteine scanning mutagenesis, we found that VDAC3's cysteine residues regulate its interaction with α-synuclein, demonstrating VDAC3-unique functional properties and further highlighting a general molecular mechanism for VDAC isoform-specific regulation of mitochondrial bioenergetics.

Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix.

Myotonia is a state of hyperexcitability of skeletal-muscle fibres. Mutations in the ClC-1 Cl- channel cause recessive and dominant forms of this disease. Mutations have been described throughout the protein-coding region, including three sequence variations (A885P, R894X and P932L) in a distal C-terminal stretch of residues [CTD (C-terminal domain) region] that are not conserved between CLC proteins. We show that surface expression of these mutants is reduced in Xenopus oocytes compared with wild-type ClC-1. Functional, biochemical and NMR spectroscopy studies revealed that the CTD region encompasses a segment conserved in most voltage-dependent CLC channels that folds with a secondary structure containing a short type II poly-proline helix. We found that the myotonia-causing mutation A885P disturbs this structure by extending the poly-proline helix. We hypothesize that this structural modification results in the observed alteration of the common gate that acts on both pores of the channel. We provide the first experimental investigation of structural changes resulting from myotonia-causing mutations.

Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel.

Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.

Pharmacogenetics of Vascular Risk Factors in Alzheimer's Disease.

Alzheimer's disease (AD) is a polygenic/complex disorder in which genomic, epigenomic, cerebrovascular, metabolic, and environmental factors converge to define a progressive neurodegenerative phenotype. Pharmacogenetics is a major determinant of therapeutic outcome in AD. Different categories of genes are potentially involved in the pharmacogenetic network responsible for drug efficacy and safety, including pathogenic, mechanistic, metabolic, transporter, and pleiotropic genes. However, most drugs exert pleiotropic effects that are promiscuously regulated for different gene products. Only 20% of the Caucasian population are extensive metabolizers for tetragenic haplotypes integrating CYP2D6-CYP2C19-CYP2C9-CYP3A4/5 variants. Patients harboring CYP-related poor (PM) and/or ultra-rapid (UM) geno-phenotypes display more irregular profiles in drug metabolism than extensive (EM) or intermediate (IM) metabolizers. Among 111 pentagenic (APOE-APOB-APOC3-CETP-LPL) haplotypes associated with lipid metabolism, carriers of the H26 haplotype (23-TT-CG-AG-CC) exhibit the lowest cholesterol levels, and patients with the H104 haplotype (44-CC-CC-AA-CC) are severely hypercholesterolemic. Furthermore, APOE, NOS3, ACE, AGT, and CYP variants influence the therapeutic response to hypotensive drugs in AD patients with hypertension. Consequently, the implementation of pharmacogenetic procedures may optimize therapeutics in AD patients under polypharmacy regimes for the treatment of concomitant vascular disorders.

Megalencephalic leukoencephalopathy with subcortical cysts: A personal biochemical retrospective.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.

Vacuolating megalencephalic leukoencephalopathy with subcortical cysts: functional studies of novel variants in MLC1.

Nine new unrelated patients presenting vacuolating myelinopathy with subcortical cysts were identified and analyzed for variations in the MLC1 gene. We detected 12 mutations (p.Leu37fs, p.Met80Val, p.Leu83Phe, p.Pro92Ser, p.Ser93Leu, p.Ile108fs, p.Gly130Arg, p.Cys171fs, p.Glu202Lys, p.Ser269Tyr, p.Ala275Asn, and p.Leu310_311insLeu) of which nine were novel. In one patient we did not detect mutations. Using a heterologous system, three new missense variants (p.Glu202Lys, p.Ser269Tyr, and p.Ala275Asn) and a single leucine insertion (p.Leu310insLeu)--lying in a stretch of seven leucines--were functionally assayed by determining total protein levels and mutant protein expression at the plasma membrane. No correlation was observed between mutation, clinical features, and plasma membrane expression of mutant protein.

Pharmacogenetic considerations in the treatment of Alzheimer's disease.

The practical pharmacogenetics of Alzheimer's disease (AD) is circumscribed to acetylcholinesterase inhibitors (AChEIs) and memantine. However, pharmacogenetic procedures should be applied to novel strategies in AD therapeutics including: novel AChEIs and neurotransmitter regulators, anti-Aß treatments, anti-tau treatments, pleiotropic products, epigenetic drugs and combination therapies. Genes involved in the pharmacogenetic network are under the influence of the epigenetic machinery which regulates gene expression transcriptionally and post-transcriptionally, configuring the fundamentals of pharmacoepigenomics. Over 60% of AD patients present concomitant pathologies demanding additional treatments which increase the likelihood of drug-drug interactions. Lipid metabolism dysfunction is a pathogenic mechanism inherent to AD neurodegeneration. The therapeutic response to hypolipidemic compounds is influenced by the APOE and CYP genotypes. The development of novel compounds and the use of combination/multifactorial treatments require the implantation of pharmacogenomic procedures for the avoidance of ADRs and the optimization of therapeutics.