Adenosine signalling to astrocytes coordinates brain metabolism and function.

Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.

Pharmacological sequestration of mitochondrial calcium uptake protects against dementia and ß-amyloid neurotoxicity.

All forms of dementia including Alzheimer's disease are currently incurable. Mitochondrial dysfunction and calcium alterations are shown to be involved in the mechanism of neurodegeneration in Alzheimer's disease. Previously we have described the ability of compound Tg-2112x to protect neurons via sequestration of mitochondrial calcium uptake and we suggest that it can also be protective against neurodegeneration and development of dementia. Using primary co-culture neurons and astrocytes we studied the effect of Tg-2112x and its derivative Tg-2113x on ß-amyloid-induced changes in calcium signal, mitochondrial membrane potential, mitochondrial calcium, and cell death. We have found that both compounds had no effect on ß-amyloid or acetylcholine-induced calcium changes in the cytosol although Tg2113x, but not Tg2112x reduced glutamate-induced calcium signal. Both compounds were able to reduce mitochondrial calcium uptake and protected cells against ß-amyloid-induced mitochondrial depolarization and cell death. Behavioral effects of Tg-2113x on learning and memory in fear conditioning were also studied in 3 mouse models of neurodegeneration: aged (16-month-old) C57Bl/6j mice, scopolamine-induced amnesia (3-month-old mice), and 9-month-old 5xFAD mice. It was found that Tg-2113x prevented age-, scopolamine- and cerebral amyloidosis-induced decrease in fear conditioning. In addition, Tg-2113x restored fear extinction of aged mice. Thus, reduction of the mitochondrial calcium uptake protects neurons and astrocytes against ß-amyloid-induced cell death and contributes to protection against dementia of different ethology. These compounds could be used as background for the developing of a novel generation of disease-modifying neuroprotective agents.

Activation of RAGE leads to the release of glutamate from astrocytes and stimulates calcium signal in neurons.

The receptor for advanced glycation end products (RAGE) is a signal receptor first shown to be activated by advanced glycation end products, but also by a variety of signal molecules, including pathological advanced oxidation protein products and ß-amyloid. However, most of the RAGE activators have multiple intracellular targets, making it difficult to unravel the exact pathway of RAGE activation. Here, we show that the cell-impermeable RAGE fragment sequence (60-76) of the V-domain of the receptor is able to activate RAGE present on the plasma membrane of neurons and, preferentially, astrocytes. This leads to the exocytosis of vesicular glutamate transporter vesicles and the release of glutamate from astrocytes, which stimulate NMDA and AMPA/kainate receptors, resulting in calcium signals predominantly in neurons. Thus, we show a specific mechanism of RAGE activation by the RAGE fragment and propose a mechanism by which RAGE activation can contribute to the neuronal-astrocytic communication in physiology and pathology.

Variability of mitochondrial energy balance across brain regions.

Brain is not homogenous and neurons from various brain regions are known to have different vulnerabilities to mitochondrial mutations and mitochondrial toxins. However, it is not clear if this vulnerability is connected to different energy metabolism in specific brain regions. Here, using live-cell imaging, we compared mitochondrial membrane potential and nicotinamide adenine dinucleotide (NADH) redox balance in acute rat brain slices in different brain regions and further detailed the mitochondrial metabolism in primary neurons and astrocytes from rat cortex, midbrain and cerebellum. We have found that mitochondrial membrane potential is higher in brain slices from the hippocampus and brain stem. In primary co-cultures, mitochondrial membrane potential in astrocytes was lower than in neurons, whereas in midbrain cells it was higher than in cortex and cerebellum. The rate of NADH production and mitochondrial NADH pool were highest in acute slices from midbrain and midbrain primary neurons and astrocytes. Although the level of adenosine tri phosphate (ATP) was similar among primary neurons and astrocytes from cortex, midbrain and cerebellum, the rate of ATP consumption was highest in midbrain cells that lead to faster neuronal and astrocytic collapse in response to inhibitors of ATP production. Thus, midbrain neurons and astrocytes have a higher metabolic rate and ATP consumption that makes them more vulnerable to energy deprivation.

Mechanosensory Signaling in Astrocytes.

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca2+ responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y1 receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca2+ responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca2+ signals via P2Y1 receptors and Ca2+ recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits.SIGNIFICANCE STATEMENT There is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca2+ responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion.

Tau inhibits mitochondrial calcium efflux and makes neurons vulnerable to calcium-induced cell death.

Aggregation or phosphorylation of the microtubule-associated protein tau is the pathological hallmark in a number of diseases termed tauopathies, which include the most common neurodegenerative disorder, Alzheimer's disease; or frontotemporal dementia, linked to mutations in the gene MAPT encoding tau. Although misfolded tau has strong familial and histopathological (as in intracellular tangles) association with neurodegenerative disorders, the cellular mechanism of tau-induced pathology remains to be controversial. Here we studied the effect of tau on the cytosolic and mitochondrial calcium homeostasis using primary cortical cultures treated with the protein and iPSC-derived neurons bearing the 10 + 16 MAPT mutation linked to frontotemporal dementia. We found that incubation of the primary cortical co-cultures of neurons and astrocytes with tau induced spontaneous Ca2+ oscillations in the neurons, which were also observed in iPSC-neurons with the 10 + 16 MAPT mutation. Importantly, tau inhibited mitochondrial calcium efflux via the mitochondrial Na+/Ca2+ exchanger (NCLX) in both neurons and astrocytes. This inhibition led to mitochondrial depolarisation in response to physiological and pathological calcium stimuli and made these cells vulnerable to calcium-induced caspase 3 activation and cell death. Thus, inhibition of the mitochondrial NCLX in neurons with misfolded or mutated tau can be involved in the mechanism of neurodegeneration.

Pathogenic p62/SQSTM1 mutations impair energy metabolism through limitation of mitochondrial substrates.

Abnormal mitochondrial function has been found in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mutations in the p62 gene (also known as SQSTM1) which encodes the p62 protein have been reported in both disorders supporting the idea of an ALS/FTD continuum. In this work the role of p62 in energy metabolism was studied in fibroblasts from FTD patients carrying two independent pathogenic mutations in the p62 gene, and in a p62-knock-down (p62 KD) human dopaminergic neuroblastoma cell line (SH-SY5Y). We found that p62 deficiency is associated with inhibited complex I mitochondrial respiration due to lack of NADH for the electron transport chain. This deficiency was also associated with increased levels of NADPH reflecting a higher activation of pentose phosphate pathway as this is accompanied with higher cytosolic reduced glutathione (GSH) levels. Complex I inhibition resulted in lower mitochondrial membrane potential and higher cytosolic ROS production. Pharmacological activation of transcription factor Nrf2 increased mitochondrial NADH levels and restored mitochondrial membrane potential in p62-deficient cells. Our results suggest that the phenotype is caused by a loss-of-function effect, because similar alterations were found both in the mutant fibroblasts and the p62 KD model. These findings highlight the implication of energy metabolism in pathophysiological events associated with p62 deficiency.

Deficiency of Parkinson's disease-related gene Fbxo7 is associated with impaired mitochondrial metabolism by PARP activation.

The Parkinson's disease (PD)-related protein F-box only protein 7 (Fbxo7) is the substrate-recognition component of the Skp1-Cullin-F-box protein E3 ubiquitin ligase complex. We have recently shown that PD-associated mutations in Fbxo7 disrupt mitochondrial autophagy (mitophagy), suggesting a role for Fbxo7 in modulating mitochondrial homeostasis. Here we report that Fbxo7 deficiency is associated with reduced cellular NAD+ levels, which results in increased mitochondrial NADH redox index and impaired activity of complex I in the electron transport chain. Under these conditions of compromised respiration, mitochondrial membrane potential and ATP contents are reduced, and cytosolic reactive oxygen species (ROS) production is increased. ROS activates poly (ADP-ribose) polymerase (PARP) activity in Fbxo7-deficient cells. PARP inhibitor restores cellular NAD+ content and redox index and ATP pool, suggesting that PARP overactivation is cause of decreased complex I-driven respiration. These findings bring new insight into the mechanism of Fbxo7 deficiency, emphasising the importance of mitochondrial dysfunction in PD.

Familial Alzheimer's Disease Lymphocytes Respond Differently Than Sporadic Cells to Oxidative Stress: Upregulated p53-p21 Signaling Linked with Presenilin 1 Mutants.

Familial (FAD) and sporadic (SAD) Alzheimer's disease do not share all pathomechanisms, but knowledge on their molecular differences is limited. We previously reported that cell cycle control distinguishes lymphocytes from SAD and FAD patients. Significant differences were found in p21 levels of SAD compared to FAD lymphocytes. Since p21 can also regulate apoptosis, the aim of this study was to compare the response of FAD and SAD lymphocytes to oxidative stress like 2-deoxy-D-ribose (2dRib) treatment and to investigate the role of p21 levels in this response. We report that FAD cells bearing seven different PS1 mutations are more resistant to 2dRib-induced cell death than control or SAD cells: FAD cells showed a lower apoptosis rate and a lower depolarization of the mitochondrial membrane. Despite that basal p21 cellular content was lower in FAD than in SAD cells, in response to 2dRib, p21 mRNA and protein levels significantly increased in FAD cells. Moreover, we found a higher cytosolic accumulation of p21 in FAD cells. The transcriptional activation of p21 was shown to be dependent on p53, as it can be blocked by PFT-α, and correlated with the increased phosphorylation of p53 at Serine 15. Our results suggest that in FAD lymphocytes, the p53-mediated increase in p21 transcription, together with a shift in the nucleocytoplasmic localization of p21, confers a survival advantage against 2dRib-induced apoptosis. This compensatory mechanism is absent in SAD cells. Thus, therapeutic and diagnostic designs should take into account possible differential apoptotic responses in SAD versus FAD cells.

Targeting cyclin D3/CDK6 activity for treatment of Parkinson's disease.

At present, treatment for Parkinson's disease (PD) is only symptomatic; therefore, it is important to identify new targets tackling the molecular causes of the disease. We previously found that lymphoblasts from sporadic PD patients display increased activity of the cyclin D3/CDK6/pRb pathway and higher proliferation than control cells. These features were considered systemic manifestations of the disease, as aberrant activation of the cell cycle is involved in neuronal apoptosis. The main goal of this work was to elucidate whether the inhibition of cyclin D3/CDK6-associated kinase activity could be useful in PD treatment. For this purpose, we investigated the effects of two histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic (SAHA) acid and sodium butyrate (NaB), and the m-TOR inhibitor rapamycin on cell viability and cyclin D3/CDK6 activity. Moreover, the potential neuroprotective action of these drugs was evaluated in 6-hydroxy-dopamine (6-OHDA) treated dopaminergic SH-SY5Y cells and primary rat mesencephalic cultures. Here, we report that both compounds normalized the proliferative activity of PD lymphoblasts and reduced the 6-OHDA-induced cell death in neuronal cells by preventing the over-activation of the cyclin D3/CDK6/pRb cascade. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it is plausible that they may serve as novel therapeutic drugs for PD. We report here that peripheral cells from Parkinson's disease (PD) patients show an enhanced proliferative activity due to the activation of cyclin D3/CDK6-mediated phosphorylation of retinoblastoma protein (pRb). Treatment of PD lymphoblasts with inhibitors of histone deacetylases like suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB), or with rapamycin, inhibitor of mechanistic target of rapamycin (mTOR) normalized the proliferation of PD lymphoblasts by preventing the over-activation of the cyclin D3/CDK6/pRb cascade. These drugs were shown to have neuroprotective effects in both human neuroblastoma SH-SY5Y cells and primary rat mid-brain dopaminergic neuronal cultures toxicity induced by 6-hidroxydopamine. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it seems reasonable to believe that the repositioning of these drugs toward PD holds promise as a novel therapeutic strategy.

Increasing progranulin levels and blockade of the ERK1/2 pathway: upstream and downstream strategies for the treatment of progranulin deficient frontotemporal dementia.

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disorder marked by mild-life onset and progressive changes in behavior, social cognition, and language. Loss-of-function progranulin gene (GRN) mutations are the major cause of FTLD with TDP-43 protein inclusions (FTLD-TDP). Disease-modifying treatments for FTLD-TDP are not available yet. Mounting evidence indicates that cell cycle dysfunction may play a pathogenic role in neurodegenerative disorders including FTLD. Since cell cycle re-entry of posmitotic neurons seems to precede neuronal death, it was hypothesized that strategies aimed at preventing cell cycle progression would have neuroprotective effects. Recent research in our laboratory revealed cell cycle alterations in lymphoblasts from FTLD-TDP patients carrying a null GRN mutation, and in PGRN deficient SH-SY5Y neuroblastoma cells, involving overactivation of the ERK1/2 signaling pathway. In this work, we have investigated the effects of PGRN enhancers drugs and ERK1/2 inhibitors, in these cellular models of PGRN-deficient FTLD. We report here that both restoring the PGRN content, by suberoylanilide hydroxamic acid (SAHA) or chloroquine (CQ), as blocking ERK1/2 activation by selumetinib (AZD6244) or MEK162 (ARRY-162), normalized the CDK6/pRb pathway and the proliferative activity of PGRN deficient cells. Moreover, we found that SAHA and selumetinib prevented the cytosolic TDP-43 accumulation in PGRN-deficient lymphoblasts. Considering that these drugs are able to cross the blood-brain barrier, and assuming that the alterations in cell cycle and signaling observed in lymphoblasts from FTLD patients could be peripheral signs of the disease, our results suggest that these treatments may serve as novel therapeutic drugs for FTLD associated to GRN mutations.

Progranulin Deficiency Reduces CDK4/6/pRb Activation and Survival of Human Neuroblastoma SH-SY5Y Cells.

Null mutations in GRN are associated with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). However, the influence of progranulin (PGRN) deficiency in neurodegeneration is largely unknown. In neuroblastoma cells, silencing of GRN gene causes significantly reduced cell survival after serum withdrawal. The following observations suggest that alterations of the CDK4/6/retinoblastoma protein (pRb) pathway, secondary to changes in PI3K/Akt and ERK1/2 activation induced by PGRN deficiency, are involved in the control of serum deprivation-induced apoptosis: (i) inhibiting CDK4/6 levels or their associated kinase activity by sodium butyrate or PD332991 sensitized control SH-SY5Y cells to serum deprivation-induced apoptosis without affecting survival of PGRN-deficient cells; (ii) CDK4/6/pRb seems to be downstream of the PI3K/Akt and ERK1/2 signaling pathways since their specific inhibitors, LY294002 and PD98059, were able to decrease CDK6-associated kinase activity and induce death of control SH-SY5Y cells; (iii) PGRN-deficient cells show reduced stimulation of PI3K/Akt, ERK1/2, and CDK4/6 activities compared with control cells in the absence of serum; and (iv) supplementation of recombinant human PGRN was able to rescue survival of PGRN-deficient cells. These observations highlight the important role of PGRN-mediated stimulation of the PI3K/Akt-ERK1/2/CDK4/6/pRb pathway in determining the cell fate survival/death under serum deprivation.

PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers.

Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Previously, we reported cell cycle-related alterations in lymphoblasts from FTLD-TDP patients, carrying the c.709-1G>A null PGRN mutation, suggesting aberrant cell cycle activation in affected neurons. Here we report that PGRN haploinsufficiency activates the extracellular signal-regulated protein kinases 1 and 2 pathway in a Ca(2+), protein kinase C-dependent, and pertussis toxin-sensitive manner. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that noncanonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II. Moreover, treatment of control cells with exogenous Wingless-type 5a (Wnt5a)-activated Ca(2+)/calmodulin kinase II (CaMKII), increased extracellular signal-regulated protein kinases 1 and 2 activity and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLD-TDP pathology and suggest a novel target for therapeutic intervention.

Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)].

Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.

Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia.

Frontotemporal lobar degeneration with neuronal inclusions containing TAR DNA binding protein 43 (TDP-43) is associated in most cases with null-mutations in the progranulin gene (PGRN). While the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration remained speculative, increasing evidence support the hypothesis that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Based in the mitogenic and neurotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability. Because cell cycle dysfunction is not restricted to neurons, we studied the influence of PGRN haploinsufficiency, on cell cycle control in peripheral cells from patients suffering from frontotemporal dementia, bearing the PGRN mutation c.709-1G>A. Here we show that progranulin deficit increased cell cycle activity in immortalized lymphocytes. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G(1)/S regulatory failure. A loss of function of TDP-43 repressing CDK6 expression may result from altered subcellular TDP-43 distribution. The distinct functional features of lymphoblastoid cells from c.709-1 G>A carriers offer an invaluable, noninvasive tool to investigate the etiopathogenesis of frontotemporal lobar degeneration.

Simvastatin overcomes the resistance to serum withdrawal-induced apoptosis of lymphocytes from Alzheimer's disease patients.

Statins may exert beneficial effects on Alzheimer's disease (AD) patients. Based on the antineoplastic and apoptotic effects of statins in a number of cell types, we hypothesized that statins may be able to protect neurons by controlling the regulation of cell cycle and/or apoptosis. A growing body of evidence indicates that neurodegeneration involves the cell-cycle activation in postmitotic neurons. Failure of cell-cycle control is not restricted to neurons in AD patients, but occurs in peripheral cells as well. For these reasons, we studied the role of simvastatin (SIM) on cell survival/death in lymphoblasts from AD patients. We report here that SIM induces apoptosis in AD lymphoblasts deprived of serum. SIM interacts with PI3K/Akt and ERK1/2 signaling pathways thereby decreasing the serum withdrawal-enhanced levels of the CDK inhibitor p21(Cip1) (p21) and restoring the vulnerability of AD cells to trophic factor deprivation.