Jorge M. A. Oliveira

Adenosine A2A Receptor Blockade Prevents Synaptotoxicity and Memory Dysfunction Caused by β-Amyloid Peptides via p38 Mitogen-Activated Protein Kinase Pathway

Alzheimers disease (AD) is characterized by memory impairment, neurochemically by accumulation of β-amyloid peptide (namely Aβ1-42) and morphologically by an initial loss of nerve terminals. Caffeine consumption prevents memory dysfunction in different models, which is mimicked by antagonists of adenosine A2A receptors (A2ARs), which are located in synapses. Thus, we now tested whether A2AR blockade prevents the early Aβ1-42-induced synaptotoxicity and memory dysfunction and what are the underlying signaling pathways. The intracerebral administration of soluble Aβ1-42 (2 nmol) in rats or mice caused, 2 weeks later, memory impairment (decreased performance in the Y-maze and object recognition tests) and a loss of nerve terminal markers (synaptophysin, SNAP-25) without overt neuronal loss, astrogliosis, or microgliosis. These were prevented by pharmacological blockade [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261); 0.05 mg · kg−1 · d−1, i.p.; for 15 d] in rats, and genetic inactivation of A2ARs in mice. Moreover, these were synaptic events since purified nerve terminals acutely exposed to Aβ1-42 (500 nm) displayed mitochondrial dysfunction, which was prevented by A2AR blockade. SCH58261 (50 nm) also prevented the initial synaptotoxicity (loss of MAP-2, synaptophysin, and SNAP-25 immunoreactivity) and subsequent loss of viability of cultured hippocampal neurons exposed to Aβ1-42 (500 nm). This A2AR-mediated control of neurotoxicity involved the control of Aβ1-42-induced p38 phosphorylation and was independent from cAMP/PKA (protein kinase A) pathway. Together, these results show that A2ARs play a crucial role in the development of Aβ-induced synaptotoxicity leading to memory dysfunction through a p38 MAPK (mitogen-activated protein kinase)-dependent pathway and provide a molecular basis for the benefits of caffeine consumption in AD.

Nature and cause of mitochondrial dysfunction in Huntington’s disease: focusing on huntingtin and the striatum

J. Neurochem. (2010) 114, 1–12.

Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission.

The leaves of Catharanthus roseus constitute the only source of the well known indolomonoterpenic alkaloids vincristine and vinblastine. In this work we studied the biological potential of the roots, which are used in several countries as decocts or hot water extracts for the treatment of a number of conditions. The aqueous extract strongly inhibited acetylcholinesterase (AchE) in an in vitro microassay, an effect ascribable mainly to serpentine (IC(50) = 0.775 microM vs physostigmine IC(50) = 6.45 microM) as assessed with the pure compound. Pure alkaloids were tested for muscarinic and nicotinic antagonism using rat ex-vivo preparations, namely, ileum and diaphragm/phrenic-nerve, respectively. Serpentine competitively blocked muscarinic receptors with a pA(2) of 5.2, whereas the precursor ajmalicine up to 80 microM was undistinguishable from control, and catharanthine exhibited an unsurmountable muscarinic antagonism at greater than 10 microM concentrations. Nicotinic receptor mediated diaphragm contractions were fully inhibited by catharanthine (IC(50) = 59.6 microM) and ajmalicine (IC(50) = 72.3 microM), in a reversible but non-competitive manner, unlike the more potent nicotinic antagonist tubocurarine (IC(50) = 0.35 microM) whose competitive blockade was overcome by a physostigmine-induced increase in acetylcholine. Serpentine up to 100 microM did not change diaphragm contractions suggesting reduced affinity for neuromuscular nicotinic receptors. Despite strong in vitro AchE inhibition, serpentine failed to restore diaphragm contractions upon submaximal tubocurarine blockade, suggesting that poor tissue penetration may prevent serpentine from inhibiting AchE in deep neuromuscular synapses in the ex-vivo preparation. To our knowledge, the present study is the first to assess the effect of C. roseus root extracts, as well as of serpentine, ajmalicine and catharanthine on AchE. The results described herein suggest that the currently overlooked C. roseus roots may constitute a promising source of compounds with pharmaceutical interest. Moreover, given serpentines potent in vitro AchE inhibitory activity and low cholinergic receptor affinity, it is conceivable that minor structural modifications may yield a potent and selective AchE inhibitor, potentially useful for the pharmacological management of conditions such as Alzheimers disease and/or myasthenia gravis.

Mitochondrial dynamics and quality control in Huntington's disease

Huntingtons disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine expansion mutations in the huntingtin protein. Despite its ubiquitous distribution, expression of mutant huntingtin (mHtt) is particularly detrimental to medium spiny neurons within the striatum. Mitochondrial dysfunction has been associated with HD pathogenesis. Here we review the current evidence for mHtt-induced abnormalities in mitochondrial dynamics and quality control, with a particular focus on brain and neuronal data pertaining to striatal vulnerability. We address mHtt effects on mitochondrial biogenesis, protein import, complex assembly, fission and fusion, mitochondrial transport, and on the degradation of damaged mitochondria via autophagy (mitophagy). For an integrated perspective on potentially converging pathogenic mechanisms, we also address impaired autophagosomal transport and abnormal mHtt proteostasis in HD.

Metabolic profiling and biological capacity of Pieris brassicae fed with kale (Brassica oleracea L. var. acephala).

Phenolic and organic acid profiles of aqueous extracts from Pieris brassicae material and the host kale (Brassica oleracea L. var. acephala) leaves were determined by HPLC/UV-DAD/MS(n)-ESI and HPLC-UV, respectively. The identified phenolics included acylated and nonacylated flavonoid glycosides, hydroxycinnamic acyl gentiobiosides, and sulphate phenolics. Kale exhibited the highest content (11g/kg lyophilized extract), while no phenolics were identified in the butterflies or exuviae. Nine different organic acids were characterized in the materials, with kale showing the highest amount (112g/kg lyophilized extract). With the exception of the exuviae extract, the rest were screened for bioactivity. Using spectrophotometric microassays, all exhibited antiradical capacity against DPPH and NO in a concentration-dependent way, whereas only kale and excrement extracts were active against superoxide. All displayed activity on intestinal smooth muscle, albeit with distinct relaxation-contraction profiles. Larvae and butterfly extracts were more efficacious for intestinal relaxation than was kale extract, whereas excrement extract evoked only contractions, thus evidencing their different compositions. Collectively, these results show that P. brassicae sequesters and metabolizes kales phenolic compounds. Moreover, the extracts bioactivities suggest that they may constitute an interesting source of bioactive compounds whose complex chemical structures preclude either synthesis or isolation.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNAPhe transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNAPhe as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNAPhe.

Lysine deacetylases and mitochondrial dynamics in neurodegeneration

Lysine acetylation is a key post-translational modification known to regulate gene transcription, signal transduction, cellular transport and metabolism. Lysine deacetylases (KDACs), including classical KDACs (a.k.a. histone deacetylases; HDACs) and sirtuins (SIRTs), are emerging therapeutic targets in neurodegeneration. Given the strong link between abnormal mitochondrial dynamics and neurodegenerative disorders (e.g. in Alzheimer, Parkinson and Huntington diseases), here we examine the evidence for KDAC-mediated regulation of mitochondrial biogenesis, fission-fusion, movement and mitophagy. Mitochondrial biogenesis regulation was reported for SIRT1, SIRT3, and class IIa KDACs, mainly via PGC-1alpha modulation. SIRT1 or SIRT3 overexpression rescued mitochondrial density and fission-fusion balance in neurodegeneration models. Mitochondrial fission decreased with pan-classical-KDAC inhibitors and increased with nicotinamide (pan-sirtuin-inhibitor/activator depending on concentration and NAD(+) conversion). Mitochondrial movement increased with HDAC6 inhibition, but this is not yet reported for the other tubulin deacetylase SIRT2. Inhibition of HDAC6 or SIRT2 was reported neuroprotective. Mitophagy is assisted by the HDAC6 ubiquitin-binding and autophagosome-lysosome fusion promoting activities, and was also associated with SIRT1 activation. In summary, KDACs can potentially modulate multiple components of mitochondrial dynamics, however, several key points require clarification. The SIRT1-biogenesis connection relies heavily in controversial caloric restriction (CR) regimes or CR-mimetic drugs, and appears cell-type dependent, recommending caution before linking SIRT1 activation with general neuroprotection. Future studies should clarify mitochondrial fission-fusion regulation by KDACs, and the interplay between HDAC6 and SIRT1 in mitophagy. Also, further studies are required to ascertain whether HDAC6 inhibition to enhance mitochondrial trafficking does not compromise autophagy or clearance of misfolded proteins in neurodegenerative disorders.

Mitochondrial bioenergetics and dynamics in Huntington’s disease: tripartite synapses and selective striatal degeneration

Preferential striatal neurodegeneration is a hallmark of Huntington’s disease (HD) pathogenesis, which has been associated with mitochondrial dysfunction. Evidence from genetic HD models suggest that mutant huntingtin (mHtt) compromises mitochondrial bioenergetics and dynamics, preventing efficient calcium handling and ATP generation in neuronal networks. Striatal neurons receive abundant glutamatergic input from the cortex, forming tripartite synapses with astrocytic partners. These are involved in bidirectional communication, play neuroprotective roles, and emerging evidence suggests that astrocyte dysfunction supports non-cell autonomous neurodegeneration. In addition to mHtt effects, inherent mitochondria vulnerability within striatal neurons and astrocytes may contribute for preferential neurodegeneration in HD. Dysfunctional astrocytic mitochondria in cortico-striatal tripartite synapses might be particularly relevant in the pathogenesis of juvenile/infantile HD, frequently associated with seizures and abnormally large mHtt polyglutamine expansions. This review discusses our work, primarily addressing in situ mitochondrial function in neurons and astrocytes, in the context of related work within the HD-mitochondria field.

Simple and reproducible HPLC-DAD-ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots.

Catharanthus roseus is one of the most important medicinal plants worldwide. The leaves of this species are the only source of the indolomonoterpenic alkaloids vincristin (leurocristine) and vinblastin (vincaleucoblastine), whose anticancer activity represents powerful therapeutics to many diseases, such as Hodgkin lymphoma. Usually, the remaining plant parts go to waste. Here we describe a phytochemical study on this species roots. Alkaloids in aqueous extracts, the usual form of consumption of this matrix, were studied using HPLC-DAD-ESI-MS/MS, which allowed the identification of 19-S-vindolinine, vindolinine, ajmalicine and an ajmalicine isomer, tabersonine, catharanthine, serpentine and a serpentine isomer. Quantification of the identified compounds revealed that serpentine and its isomer were predominant (64.7%) over the other alkaloids, namely vindolinine and its isomer (23.9%), catharanthine (7.7%) and ajmalicine (3.8%). The used procedure revealed to be simple, sensitive and reproducible.

How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs

Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone.