Deficiency of Mitochondrial Aspartate-Glutamate Carrier 1 Leads to Oligodendrocyte Precursor Cell Proliferation Defects Both In Vitro and In Vivo.

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate-aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor α (PDGFα) and Transforming Growth Factor ßs (TGFßs). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency.

Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells.

Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)-nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10 µM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y1 or adenosine A2A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2 h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2 h cell re-oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re-oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end-products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

MRI-guided histology of TDP-43 knock-in mice implicates parvalbumin interneuron loss, impaired neurogenesis and aberrant neurodevelopment in amyotrophic lateral sclerosis-frontotemporal dementia.

Amyotrophic lateral sclerosis and frontotemporal dementia are overlapping diseases in which MRI reveals brain structural changes in advance of symptom onset. Recapitulating these changes in preclinical models would help to improve our understanding of the molecular causes underlying regionally selective brain atrophy in early disease. We therefore investigated the translational potential of the TDP-43Q331K knock-in mouse model of amyotrophic lateral sclerosis-frontotemporal dementia using MRI. We performed in vivo MRI of TDP-43Q331K knock-in mice. Regions of significant volume change were chosen for post-mortem brain tissue analyses. Ex vivo computed tomography was performed to investigate skull shape. Parvalbumin neuron density was quantified in post-mortem amyotrophic lateral sclerosis frontal cortex. Adult mutants demonstrated parenchymal volume reductions affecting the frontal lobe and entorhinal cortex in a manner reminiscent of amyotrophic lateral sclerosis-frontotemporal dementia. Subcortical, cerebellar and brain stem regions were also affected in line with observations in pre-symptomatic carriers of mutations in C9orf72, the commonest genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Volume loss was also observed in the dentate gyrus of the hippocampus, along with ventricular enlargement. Immunohistochemistry revealed reduced parvalbumin interneurons as a potential cellular correlate of MRI changes in mutant mice. By contrast, microglia was in a disease activated state even in the absence of brain volume loss. A reduction in immature neurons was found in the dentate gyrus, indicative of impaired adult neurogenesis, while a paucity of parvalbumin interneurons in P14 mutant mice suggests that TDP-43Q331K disrupts neurodevelopment. Computerized tomography imaging showed altered skull morphology in mutants, further suggesting a role for TDP-43Q331K in development. Finally, analysis of human post-mortem brains confirmed a paucity of parvalbumin interneurons in the prefrontal cortex in sporadic amyotrophic lateral sclerosis and amyotrophic lateral sclerosis linked to C9orf72 mutations. Regional brain MRI changes seen in human amyotrophic lateral sclerosis-frontotemporal dementia are recapitulated in TDP-43Q331K knock-in mice. By marrying in vivo imaging with targeted histology, we can unravel cellular and molecular processes underlying selective brain vulnerability in human disease. As well as helping to understand the earliest causes of disease, our MRI and histological markers will be valuable in assessing the efficacy of putative therapeutics in TDP-43Q331K knock-in mice.

A Focused Library of Psychotropic Analogues with Neuroprotective and Neuroregenerative Potential.

Overcoming the lack of effective treatments and the continuous clinical trial failures in neurodegenerative drug discovery might require a shift from the prevailing paradigm targeting pathogenesis to the one targeting simultaneously neuroprotection and neuroregeneration. In the studies reported herein, we sought to identify small molecules that might exert neuroprotective and neuroregenerative potential as tools against neurodegenerative diseases. In doing so, we started from the reported neuroprotective/neuroregenerative mechanisms of psychotropic drugs featuring a tricyclic alkylamine scaffold. Thus, we designed a focused-chemical library of 36 entries aimed at exploring the structural requirements for efficient neuroprotective/neuroregenerative cellular activity, without the manifestation of toxicity. To this aim, we developed a synthetic protocol, which overcame the limited applicability of previously reported procedures. Next, we evaluated the synthesized compounds through a phenotypic screening pipeline, based on primary neuronal systems. Phenothiazine 2Bc showed improved neuroregenerative and neuroprotective properties with respect to reference drug desipramine (2Aa). Importantly, we have also shown that 2Bc outperformed currently available drugs in cell models of Alzheimer's and Parkinson's diseases and attenuates microglial activation by reducing iNOS expression.

Release of soluble and vesicular purine nucleoside phosphorylase from rat astrocytes and microglia induced by pro-inflammatory stimulation with extracellular ATP via P2X7 receptors.

Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1ß (IL-1ß) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1ß secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X7 receptor (P2X7R), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2X7R, and blocked by cell pre-treatment with the P2X7R antagonist A438079. Interestingly, ATP-induced PNP release from glial cells partly occurred through the secretion of lysosomal vesicles in the extracellular medium. Thus, during inflammatory cerebral events PNP secretion promoted by extracellular ATP accumulation might concur to control extracellular purine signals. Further studies could elucidate whether, in these conditions, a consensual activity of enzymes downstream of PNP in the purine metabolic cascade avoids accumulation of extracellular purine bases that might concur to brain injury by unusual formation of reactive oxygen species.

New tacrine dimers with antioxidant linkers as dual drugs: Anti-Alzheimer's and antiproliferative agents.

We have designed a series of tacrine-based homo- and heterodimers that incorporate an antioxidant tether (selenoureido, chalcogenide) as new dual compounds: for the treatment of Alzheimer's disease and as antiproliferative agents. Symmetrical homodimers bearing a dichalcogenide or selenide-based tether, the best compounds in the series, were found to be strong and highly selective electric eel AChE inhibitors, with inhibition constants within the low nanomolar range. This high inhibitory activity was confirmed on recombinant human AChE for the most interesting derivatives. The three most promising homodimers also showed a good inhibitory activity towards amyloid-ß self aggregation. The symmetric disulfide derivative bis[5-(1',2',3',4'-tetrahydroacridin-9'-ylamino)pentyl]disulfide (19) showed the best multipotent profile and was not neurotoxic on immortalized mouse cortical neurons even at 50 µM concentration. These results represent an improvement in activity and selectivity compared to parent tacrine, the first marketed drug against Alzheimer's disease. Title compounds also exhibited excellent in vitro antiproliferative activities against a panel of 6 human tumor cell lines, with GI50 values within the submicromolar range for the most potent derivatives (0.12-0.95 µM); such values represent a spectacular increase compared to currently-used chemotherapeutic agents, such as 5-FU (up to 306-fold) and cisplatin (up to 162-fold). Cell cycle experiments indicated the accumulation of cells in the G1 phase of the cycle, a different mechanism than the reported for cisplatin. The breast cancer cell lines turned out to be the most sensitive one of the panel tested.

Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease.

Multi-target drug discovery is one of the most followed approaches in the active central nervous system (CNS) therapeutic area, especially in the search for new drugs against Alzheimer's disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Aß self-aggregation in vitro. In addition, 12 showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.

Multitarget drug design strategy: quinone-tacrine hybrids designed to block amyloid-ß aggregation and to exert anticholinesterase and antioxidant effects.

We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-ß (Aß) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Aß. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.

Histone post-translational modifications in Huntington's and Parkinson's diseases.

Gene expression is controlled by several epigenetic mechanisms involving post-translational modification of histones (acetylation, phosphorylation and others). These mechanisms in the brain are not only important for normal function but also for the development of pathologies when their derangement does occur. The present review deals with post-translational modifications of histones in two neurodegenerative diseases characterized by different etiology and pathological progression, Huntington's disease and Parkinson's disease. A relatively large body of evidence supports an important role of these mechanisms in Huntington's disease while knowledge of similar mechanisms in Parkinson's disease is at a lower degree of understanding. Starting from available information on pathologies, the present state of possible therapeutic targets is considered and future developments are discussed.