Juan A. Parga

Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons

Cell death induced by 6‐hydroxydopamine (6‐OHDA) is thought to be caused by reactive oxygen species (ROS) derived from 6‐OHDA autooxidation and by a possible direct effect of 6‐OHDA on the mitochondrial respiratory chain. However, the process has not been totally clarified. In rat primary mesencephalic cultures, we observed a significant increase in dopaminergic (DA) cell loss 24 h after administration of 6‐OHDA (40 μmol/L) and a significant increase in NADPH subunit expression, microglial activation and superoxide anion/superoxide‐derived ROS in DA cells that were decreased by the NADPH inhibitor apocynin. Low doses of 6‐OHDA (10 μmol/L) did not induce a significant loss of DA cells or a significant increase in NADPH subunit expression, microglial activation or superoxide‐derived ROS. However, treatment with the NADPH complex activator angiotensin II caused a significant increase in all the latter. Forty‐eight hours after intrastriatal 6‐OHDA injection in rats, there was still no loss of DA neurons although there was an increase in NADPH subunit expression and NADPH oxidase activity. The results suggest that in addition to the autooxidation‐derived ROS and the inhibition of the mitochondrial respiratory chain, early microglial activation and NADPH oxidase‐derived ROS act synergistically with 6‐OHDA and constitute a relevant and early component of the 6‐OHDA‐induced cell death.

Brain angiotensin enhances dopaminergic cell death via microglial activation and NADPH-derived ROS

Angiotensin II (AII) plays a major role in the progression of inflammation and NADPH-derived oxidative stress (OS) in several tissues. The brain possesses a local angiotensin system, and OS and inflammation are key factors in the progression of Parkinsons disease. In rat mesencephalic cultures, AII increased 6-OHDA-induced dopaminergic (DA) cell death, generation of superoxide in DA neurons and microglial cells, the expression of NADPH-oxidase mRNA, and the number of reactive microglial cells. These effects were blocked by AII type-1 (AT1) antagonists, NADPH inhibitors, or elimination of glial cells. DA degeneration increased angiotensin converting enzyme activity and AII levels. In rats, 6-OHDA-induced dopaminergic cell loss and microglial activation were reduced by treatment with AT1 antagonists. The present data suggest that AII, via AT1 receptors, increases the dopaminergic degeneration process by amplifying the inflammatory response and intraneuronal levels of OS, and that glial cells play a major role in this process.

Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.

Angiotensin II increases differentiation of dopaminergic neurons from mesencephalic precursors via angiotensin type 2 receptors.

In addition to the well‐known actions of the humoral renin–angiotensin system, all components of this system are present in many tissues, including the brain, and may play a major role in brain development and differentiation. We investigated the possible effects of angiotensin II on the generation of dopaminergic phenotype neurons from proliferating neurospheres of mesencephalic precursors. We observed immunoreactivity for both angiotensin type 1 and type 2 (AT1 and AT2) receptors in the cell aggregates. Double immunolabeling studies revealed that both receptor types are located in neurons and astrocytes. Interestingly, neurons with a dopaminergic phenotype (i.e. tyrosine hydroxylase activity) showed double labeling for AT1 and AT2 receptors although the labeling for AT2 was more intense. Treatment of the neurospheres with angiotensin II (100 nm) during the differentiation period induced a marked increase (about 400%) in the generation of dopaminergic neurons. This was not affected by treatment with the AT1 antagonist ZD 7155 but was blocked by treatment with the AT2 antagonist PD 123319. This suggests that AT2 receptors mediate the stimulatory effect of angiotensin II on the generation of dopaminergic neurons. Apoptotic cell death studies and bromodeoxyuridine immunohistochemistry indicated that the increase in generation of dopaminergic neurons is not due to increased survival or proliferation of dopaminergic cells during treatment with angiotensin and suggested that angiotensin induces increased differentiation of mesencephalic precursors towards the dopaminergic phenotype. Manipulation of the renin–angiotensin system may be useful for increasing production of dopaminergic neurons for transplantation in Parkinsons disease.

Discovery of Inhibitors of Microglial Neurotoxicity Acting Through Multiple Mechanisms Using a Stem-Cell-Based Phenotypic Assay

Stem cells, through their ability to both self-renew and differentiate, can produce a virtually limitless supply of specialized cells that behave comparably to primary cells. We took advantage of this property to develop an assay for small-molecule-based neuroprotection using stem-cell-derived motor neurons and astrocytes, together with activated microglia as a stress paradigm. Here, we report on the discovery of hit compounds from a screen of more than 10,000 small molecules. These compounds act through diverse pathways, including the inhibition of nitric oxide production by microglia, activation of the Nrf2 pathway in microglia and astrocytes, and direct protection of neurons from nitric-oxide-induced degeneration. We confirm the activity of these compounds using human neurons. Because microglial cells are activated in many neurological disorders, our hit compounds could be ideal starting points for the development of new drugs to treat various neurodegenerative and neurological diseases.

Highly Enantioselective Catalytic Synthesis of Neurite Growth-Promoting Secoyohimbanes

Natural products endowed with neuromodulatory activity and their underlying structural scaffolds may inspire the synthesis of novel neurotrophic compound classes. The spirocyclic secoyohimbane alkaloid rhynchophylline is the major component of the extracts of Uncaria species used in Chinese traditional medicine for treatment of disorders of the central nervous system. Based on the structure of rhynchophylline, a highly enantioselective and efficient organocatalyzed synthesis method was developed that gives access to the tetracyclic secoyohimbane scaffold, embodying a quaternary and three tertiary stereogenic centers in a one-pot multistep reaction sequence. Investigation of a collection of the secoyohimbanes in primary rat hippocampal neurons and embryonal stem cell-derived motor neurons led to discovery of compounds that promote neurite outgrowth and influence the complexity of neuronal network formation.

The Mitochondrial ATP-Sensitive Potassium Channel Blocker 5-Hydroxydecanoate Inhibits Toxicity of 6-Hydroxydopamine on Dopaminergic Neurons

The neurotoxin 6-hydroxydopamine is commonly used in models of Parkinson’s disease, and a potential factor in the pathogenesis of the disease. However, the mechanisms responsible for 6-hydroxydopamine-induced dopaminergic degeneration have not been totally clarified. Reactive oxygen species (ROS) derived from 6-OHDA uptake and intraneuronal autooxidation, extracellular 6-OHDA autooxidation, and microglial activation have been involved. The mitochondrial implication is controversial. Mitochondrial ATP-sensitive K (mitoK(ATP)) channels may provide a convergent target that could integrate these different mechanisms. We observed that in primary mesencephalic cultures and neuron-enriched cultures, treatment with the mitoK(ATP) channel blocker 5-hydroxydecanoate, inhibits the dopaminergic degeneration induced by low doses of 6-OHDA. Furthermore, 5-hydroxydecanoate blocks the 6-OHDA-induced decrease in mitochondrial inner membrane potential and inhibits 6-OHDA-induced generation of superoxide-derived ROS in dopaminergic neurons. The results suggest that low doses of 6-OHDA may generate low levels of ROS through several mechanisms, which may be insufficient to induce neuron death. However, they could act as a trigger to activate mitoK(ATP) channels, thereby enhancing ROS production and the subsequent dopaminergic degeneration. Furthermore, the present study provides additional data for considering mitoK(ATP) channels as a potential target for neuroprotection.

Mitochondrial ATP-sensitive potassium channels enhance angiotensin-induced oxidative damage and dopaminergic neuron degeneration. Relevance for aging-associated susceptibility to Parkinson's disease.

Recent studies have shown that renin–angiotensin system overactivation is involved in the aging process in several tissues as well as in longevity and aging-related degenerative diseases by increasing oxidative damage and inflammation. We have recently shown that angiotensin II enhances dopaminergic degeneration by increasing levels of reactive oxygen species and neuroinflammation, and that there is an aging-related increase in angiotensin II activity in the substantia nigra in rats, which may constitute a major factor in the increased risk of Parkinson’s disease with aging. The mechanisms involved in the above mentioned effects and particularly a potential angiotensin–mitochondria interaction have not been clarified. The present study revealed that activation of mitochondrial ATP-sensitive potassium channels [mitoK(ATP)] may play a major role in the angiotensin II-induced effects on aging and neurodegeneration. Inhibition of mitoK(ATP) channels with 5-hydroxydecanoic acid inhibited the increase in dopaminergic cell death induced by angiotensin II, as well as the increase in superoxide/superoxide-derived reactive oxygen species levels and the angiotensin II-induced decrease in the mitochondrial inner membrane potential in cultured dopaminergic neurons. The present study provides data for considering brain renin–angiotensin system and mitoK(ATP) channels as potential targets for protective therapy in aging-associated diseases such as Parkinson’s disease.

Glial overexpression of heme oxygenase-1: a histochemical marker for early stages of striatal damage.

The level of heme oxygenase-1 (HO-1) in the normal striatum is below the limit of immunodetection. However, HO-1 is overexpressed in both neural and non-neural cells in response to a wide range of lesions. We induced different types of lesions affecting the striatal cells or the main striatal afferent systems in rats to investigate if overexpression of HO-1 could be a useful histochemical marker of striatal damage. Thirty-six hours after intrastriatal or intraventricular injection of excitotoxins that affect striatal neurons (ibotenic acid) or of neurotoxins that affect striatal dopaminergic (6-hydroxydopamine) or serotonergic (5,7-dihydroxytriptamine) afferent terminals, or after surgical lesioning of cortico-striatal projections, there was intense induction of striatal HO-1 immunoreactivity (HO-1-ir). Double immunolabeling revealed that the HO-1-ir was located in glial cells. After intrastriatal injection of ibotenic acid, a central zone of neuronal degeneration contained numerous round and pseudopodic HO-1-ir cells, and was surrounded by a ring of HO-1-ir cells, most of which were immunoreactive for astroglial markers. Intraventricular injection of neurotoxins induced astroglial HO-1-ir cells which were more evenly distributed throughout the lesioned or denervated areas. HO-1-ir microglial cells were also observed in areas subjected to mechanical damage. The HO-1-ir was markedly lower or absent 1 week after lesion, and even more so 3 weeks after, although some HO-1-ir cells were still observed after intrastriatal injection of ibotenic acid or surgical corticostriatal deafferentation. The results indicate that determination of glial HO-1-ir is a useful histochemical marker for early stages of striatal damage.

Paracrine and Intracrine Angiotensin 1-7/Mas Receptor Axis in the Substantia Nigra of Rodents, Monkeys, and Humans

In addition to the classical hormonal (tissue-to-tissue) renin-angiotensin system (RAS), there are a paracrine (cell-to-cell) and an intracrine (intracellular/nuclear) RAS. A local paracrine brain RAS has been associated with several brain disorders, including Parkinson’s disease (PD). Classically, angiotensin II (Ang II) is the main RAS effector peptide and acts through two major receptors: Ang II type 1 and 2 (AT1 and AT2) receptors. It has been shown that enhanced activation of the Ang II/AT1 axis exacerbates dopaminergic cell death. Several new components of the RAS have more recently been discovered. However, the role of new Ang 1-7/Mas receptor RAS component was not investigated in the brain and particularly in the dopaminergic system. In the present study, we observed Mas receptor labeling in dopaminergic neurons and glial cells in rat mesencephalic primary cultures; substantia nigra of rats, monkeys, and humans; and human induced pluripotent stem (iPS) cells derived from healthy controls and sporadic PD patients. The present data support a neuroprotective role of the Ang 1-7/Mas receptor axis in the dopaminergic system. We observed that this axis is downregulated with aging, which may contribute to the aging-related vulnerability to neurodegeneration. We have also identified an intracellular Ang 1-7/Mas axis that modulates mitochondrial and nuclear levels of superoxide. The present data suggest that nuclear RAS receptors regulate the adequate balance between the detrimental and the protective arms of the cell RAS. The results further support that the brain RAS should be taken into account for the design of new therapeutic strategies for PD.