Cerebrospinal Fluid Mitochondrial DNA in Rapid and Slow Progressive Forms of Alzheimer's Disease

Alzheimer's type dementia (AD) exhibits clinical heterogeneity, as well as differences in disease progression, as a subset of patients with a clinical diagnosis of AD progresses more rapidly (rpAD) than the typical AD of slow progression (spAD). Previous findings indicate that low cerebrospinal fluid (CSF) content of cell-free mitochondrial DNA (cf-mtDNA) precedes clinical signs of AD. We have now investigated the relationship between cf-mtDNA and other biomarkers of AD to determine whether a particular biomarker profile underlies the different rates of AD progression. We measured the content of cf-mtDNA, beta-amyloid peptide 1-42 (A beta), total tau protein (t-tau) and phosphorylated tau (p-tau) in the CSF from a cohort of 95 subjects consisting of 49 controls with a neurologic disorder without dementia, 30 patients with a clinical diagnosis of spAD and 16 patients with rpAD. We found that 37% of controls met at least one AD biomarker criteria, while 53% and 44% of subjects with spAD and rpAD, respectively, did not fulfill the two core AD biomarker criteria: high t-tau and low A beta in CSF. In the whole cohort, patients with spAD, but not with rpAD, showed a statistically significant 44% decrease of cf-mtDNA in CSF compared to control. When the cohort included only subjects selected by A beta and t-tau biomarker criteria, the spAD group showed a larger decrease of cf-mtDNA (69%), whereas in the rpAD group cf-mtDNA levels remained unaltered. In the whole cohort, the CSF levels of cf-mtDNA correlated positively with A beta and negatively with p-tau. Moreover, the ratio between cf-mtDNA and p-tau increased the sensitivity and specificity of spAD diagnosis up to 93% and 94%, respectively, in the biomarker-selected cohort. These results show that the content of cf-mtDNA in CSF correlates with the earliest pathological markers of the disease, A beta and p-tau, but not with the marker of neuronal damage t-tau. Moreover, these findings confirm that low CSF content of cf-mtDNA is a biomarker for the early detection of AD and support the hypothesis that low cf-mtDNA, together with low A beta and high p-tau, constitute a distinctive CSF biomarker profile that differentiates spAD from other neurological disorders

Molecular Basis for Maternal Inheritance of Human Mitochondrial DNA

Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization1, 2. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy3, 4. However, whether these mechanisms play a decisive role in paternal mtDNA elimination has been disputed5, 6. We found that mitochondria in human spermatozoa are devoid of mtDNA and lack mitochondrial transcription factor A (TFAM), the major nucleoid protein required to protect, maintain, and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial pre-sequence, ordinarily removed upon mitochondrial import. Phosphorylation of this pre-sequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM re-localization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mitochondrial DNA, thereby explaining maternal inheritance in this species.National Institutes of Health grant R35 GM131832 (DT). PID2020-115091RB-I00, MCIN/AEI/10.13039/501100011033 Spain (RT). PI2020/09-4, CIBERNED, Instituto de Salud Carlos III (ISCIII) Spain (RT).N

Screening for drug that increase the copy number of mitochondrial DNA

Peer Reviewe

Familial Alzheimer's disease gene mutations regulate mitochondrial DNA replication, transcription and release

Trabajo presentado en la 16th International Conference on Alzheimer's and Parkinson's Diseases (AD/PD), celebrada en Barcelona (España), del 15 al 20 de marzo de 202

Accumulation of mitochondrial 7S DNA in idiopathic and LRRK2 associated Parkinson's disease

Background: Both idiopathic and familial Parkinson's disease are associated with mitochondrial dysfunction. Mitochondria have their own mitochondrial DNA (mtDNA) and previous studies have reported that the release of mtDNA is a biomarker of Parkinson's disease. Methods: We have now investigated the relationship between mtDNA replication, transcription and release in fibroblasts from patients with idiopathic (iPD) and Leucine-rich repeat kinase 2 -associated Parkinson's disease (LRRK2-PD), using Selfie-digital PCR, a method that allows absolute quantification of mtDNA genomes and transcripts. Findings: In comparison with healthy controls, we found that fibroblasts from patients with iPD or LRRK2-PD had a high amount of mitochondrial 7S DNA along with a low mtDNA replication rate that was associated with a reduction of cf-mtDNA release. Accumulation of 7S DNA in iPD and LRRK2-PD fibroblasts was related with an increase in H-strand mtDNA transcription. Interpretation: These results show that 7S DNA accumulation, low mtDNA replication, high H-strand transcription, and low mtDNA release compose a pattern of mtDNA dysfunction shared by both iPD and LRRK2-PD fibroblasts. Moreover, these results suggest that the deregulation of the genetic switch formed by 7SDNA that alternates between mtDNA replication and transcription is a fundamental pathophysiological mechanism in both idiopathic and monogenic Parkinson's disease.This work was supported by grants to Ramon Trullas and Petar Podlesniy from the Ministerio de Ciencia, Innovación y Universidades of Spain (Grant: SAF2017-89791-R) and from the Instituto Carlos III, Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, CIBERNED (Grant: PI2016/06-3)

Accumulation of mitocondrial 7S DNA in idiopathic and LRRK2 associated Parkinson's disease

Trabajo presentado en el , celebrado en Santiago de Compostela (España), del 4 al 6 de septiembre de 201

Ca2+-modulated photoactivatable imaging reveals neuron-astrocyte glutamatergic circuitries within the nucleus accumbens

We thank Alfonso Araque, Eduardo Martín, Juliana M Rosa, and Gertrudis Perea for expert advice and critical reading of the manuscript. This work was supported by grants from the Spanish Ministry of Science and Innovation (Ramón y Cajal RYC-2016-20414, RTI2018-094887-B-I00, and PID2021-122586NB-I00) to M.N. and Fondo Europeo de Desarrollo Regional (FEDER) and PID2020-115091RB-10 to R.T. The professional editing service NB Revisions was used for technical preparation of the text prior to submission

Enhanced oxidative stress-regulated by cholesterol promotes necroptosis in Alzheimer's disease

Trabajo presentado en el Global summit on Neurodegeneratice Disease Neuro, celebrado en Salamanca (España), del 21 al 24 de junio de 2022How do neurons die in Alzheimer¿s disease (AD)? The answer is not clear. Caspases, primary effectors during apoptosis, are activated in AD brains; however, apoptotic morphology is not evident. Furthermore, the progress of the disease that lasts for decades seems incompatible with an apoptotic death program. A recent study offers compelling evidence of active necroptosis, a regulated inflammatory cell death, in post-mortem AD brains, but still, the events that trigger or regulate necroptosis in AD are not fully known. It has been reported that oxidative stress may be determinant as to whether a cell initiates necroptosis; it is, therefore, likely that mitochondrial dysfunction, observed in the early stages of AD, might favor cells to undergo necroptosis. Previous studies from our group have shown that high intracellular cholesterol levels deplete the mitochondrial pool of GSH, thus sensitizing neurons against amyloid-beta (A¿)-induced mitochondrial oxidative stress. The present study is aimed to evaluate whether cholesterol can regulate the necroptotic pathway using both APP-PSEN1 mice that overexpress SREBF2 and cholesterol-enriched SH-SY5Y cells. Methods: Cholesterol enrichment was assessed by incubating the cells with soluble cholesterol:methyl-cyclodextrin complex (50 ¿g/ml) for 1 h followed by 4 h recovery. To induce necroptosis, cells were treated for 24 h with TLQ [TNF¿ (10 ng/ml) plus the SMAC mimetic (LCL-161, 10 ¿M) and the pan-caspase inhibitor qVD-OPH (10 ¿M)]. Results: In the brains of APP-PSEN1-SREBF2 mice, we found an up-regulated expression of the necroptosis-related proteins RIPK3 and MLKL, which together with RIPK1 accumulated in the urea-soluble protein fraction, indicative of necrosome assembly. Moreover, western blot analyses of brain extracts from triple transgenic mice also showed high levels of c-FLIP, the natural inhibitor of caspase-8, further pointing to a cholesterol-regulated engagement of the necroptotic pathway. In SH-SY5Y cells, cholesterol enrichment resulted in enhanced cell death after exposure to TLQ, which was prevented by RIPK1 and RIPK3 inhibitors (Necrostatin and GSK¿872, respectively). Similar protection against TLQ-induced necroptosis was achieved when mitochondrial GSH levels were recovered by GSH ethyl ester. Intriguingly, cholesterol also regulated the intracellular localization of RIPK3 and MLKL. Confocal microscopy showed an increased presence of nuclear RIPK3 and MLKL in cholesterol-enriched cells, recently described as a requirement for the subsequent necrosome formation in the cytosol. Overall, these findings indicate that high intracellular cholesterol levels compromise neuronal viability, promoting necroptosis and subsequent necroptosis, a pro-inflammatory type of cell death, which ultimately may contribute to chronic neuroinflammation in AD.Proyectos RTI2018-095572-B-100 y PID2020-115091RB-I00 financiados por MCIN/AEI /10.13039/501100011033 y por la Unión Europea Next GenerationEU/ PRT

Enhanced oxidative stress regulated by cholesterol promotes Necroptosis in Alzheimer's disease

Trabajo presentado en la Alzheimer's Association International Conference (AAIC), celebrada en Philadelphia (Estados Unidos), del 28 de julio al 1 de agosto de 2023Background: Necroptosis has been reported in post-mortem Alzheimer’s disease (AD) brains, but still, the events that trigger or regulate this type of inflammatory cell death in AD are not fully known. Oxidative stress may be determinant as to whether a cell initiates necroptosis; Therefore, it is, likely that mitochondrial dysfunction, observed in the early stages of AD, might favor cells to undergo necroptosis. Previous studies from our group have shown that high intracellular cholesterol levels deplete the mitochondrial pool of GSH, thus sensitizing neurons against amyloid-beta (Ab)-induced mitochondrial oxidative stress. The present study is aimed to evaluate whether cholesterol can regulate the necroptotic pathway using both APP-PSEN1 mice that overexpress the cholesterol-related transcription factor SREBF2 and cholesterol-enriched SH-SY5Y cells. Method: Cholesterol enrichment was assessed by incubating the cells with soluble cholesterol:methyl-cyclodextrin complex (50 mg/ml) for 1 h followed by 4 h recovery. To induce necroptosis, cells were treated with TLQ [TNFa (10 ng/ml) plus the SMAC mimetic (LCL-161, 10 mM) and the pan-caspase inhibitor qVD-OPH (10 mM)] for 24 h. Results: Brains from APP-PSEN1-SREBF2 mice show up-regulated expression of the necroptosis-related proteins RIPK3 and MLKL, which together with RIPK1 accumulated in the urea-soluble protein fraction, indicative of necrosome assembly. Moreover, western blot analyses of brain extracts from triple transgenic mice also showed high levels of c-FLIP, the natural inhibitor of caspase-8, further pointing to a cholesterol-regulated engagement of the necroptotic pathway. In SH-SY5Y cells, cholesterol enrichment resulted in enhanced cell death after exposure to TLQ, which was prevented by RIPK1 and RIPK3 inhibitors (Necrostatin and GSK’872, respectively). Similar protection against TLQ-induced necroptosis was achieved when mitochondrial GSH levels were recovered by GSH ethyl ester. Intriguingly, cholesterol also regulated the intracellular localization of RIPK3 and MLKL. Confocal microscopy showed an increased presence of nuclear RIPK3 and MLKL in cholesterol-enriched cells, recently described as a requirement for the subsequent necrosome formation in the cytosol. Conclusion: Overall, these findings indicate that high intracellular cholesterol levels compromise neuronal viability, promoting oxidative stress and subsequent necroptosis, a pro-inflammatory type of cell death, which ultimately may contribute to chronic neuroinflammation in AD.This work was supported by grants from Ministerio de Ciencia y Innovación (RTI 2018 095572 B 100 and PID 2020 115091 RB 100 AGAUR (SGR 00490 and 2021 FI_B 0062