Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid ß aggregates in vivo in a mouse model of Alzheimer's disease.

BACKGROUND: Reactive oxidative stress is a critical player in the amyloid beta (Aß) toxicity that contributes to neurodegeneration in Alzheimer's disease (AD). Damaged mitochondria are one of the main sources of reactive oxygen species and accumulate in Aß plaque-associated dystrophic neurites in the AD brain. Although Aß causes neuronal mitochondria reactive oxidative stress in vitro, this has never been directly observed in vivo in the living mouse brain. Here, we tested for the first time whether Aß plaques and soluble Aß oligomers induce mitochondrial oxidative stress in surrounding neurons in vivo, and whether this neurotoxic effect can be abrogated using mitochondrial-targeted antioxidants. METHODS: We expressed a genetically encoded fluorescent ratiometric mitochondria-targeted reporter of oxidative stress in mouse models of the disease and performed intravital multiphoton microscopy of neuronal mitochondria and Aß plaques. RESULTS: For the first time, we demonstrated by direct observation in the living mouse brain exacerbated mitochondrial oxidative stress in neurons after both Aß plaque deposition and direct application of soluble oligomeric Aß onto the brain, and determined the most likely pathological sequence of events leading to oxidative stress in vivo. Oxidative stress could be inhibited by both blocking calcium influx into mitochondria and treating with the mitochondria-targeted antioxidant SS31. Remarkably, the latter ameliorated plaque-associated dystrophic neurites without impacting Aß plaque burden. CONCLUSIONS: Considering these results, combination of mitochondria-targeted compounds with other anti-amyloid beta or anti-tau therapies hold promise as neuroprotective drugs for the prevention and/or treatment of AD.

Near-infrared fluorescence lifetime imaging of amyloid-ß aggregates and tau fibrils through the intact skull of mice.

Non-invasive methods for the in vivo detection of hallmarks of Alzheimer's disease can facilitate the study of the progression of the disease in mouse models and may enable its earlier diagnosis in humans. Here we show that the zwitterionic heptamethine fluorophore ZW800-1C, which has peak excitation and emission wavelengths in the near-infrared optical window, binds in vivo and at high contrast to amyloid-ß deposits and to neurofibrillary tangles, and allows for the microscopic imaging of amyloid-ß and tau aggregates through the intact skull of mice. In transgenic mouse models of Alzheimer's disease, we compare the performance of ZW800-1C with that of the two spectrally similar heptamethine fluorophores ZW800-1A and indocyanine green, and show that ZW800-1C undergoes a longer fluorescence-lifetime shift when bound to amyloid-ß and tau aggregates than when circulating in blood vessels. ZW800-1C may prove advantageous for tracking the proteinic aggregates in rodent models of amyloid-ß and tau pathologies.

Low Incidence of Acute Antibody-Mediated Rejection after HLA Desensitization in Living Donor Kidney Transplant Recipients.

Desensitization allows the performance of human leukocyte antigen (HLA)-incompatible transplants. However, the incidence of acute rejection (AR) is high. This study aims to analyze the incidence of AR after transplantation with HLA-incompatible living donors in patients who underwent desensitization. Patients were immunosuppressed with tacrolimus, mycophenolic acid derivatives, and steroids after being desensitized with rituximab, plasma exchange, and/or immunoadsorption with specific cytomegalovirus immunoglobulins. A negative complement-dependent cytotoxicity or flow cytometry crossmatch and a donor-specific antibody titer < 1000 mean fluorescence intensity (MFI) were used to determine desensitization efficacy. A total of 36 patients underwent desensitization, and 27 (75%) were transplanted. After a follow-up of 58 ± 58 months (Min−Max: 0.13−169.5), five episodes of AR occurred: two antibody-mediated and three T-cell-mediated. No differences were found in baseline calculated panel-reactive antibodies (cPRA), class I or II MFI, number of antibodies, or Relative Intensity Scale (RIS) between AR and non-AR patients. Patients with antibody-mediated AR had higher cPRA (NS), MFI class I (p = 0.07) and class II (p = 0.006), and RIS (p = 0.01). The two patients with antibody-mediated AR and one patient with T-cell-mediated AR lost their grafts. In conclusion, the incidence of acute antibody-mediated rejection after desensitization was 7.4%, which occurred early post-transplantation in patients with high MFI and was associated with early graft loss.

Accelerated amyloid angiopathy and related vascular alterations in a mixed murine model of Alzheimer´s disease and type two diabetes.

BACKGROUND: While aging is the main risk factor for Alzheimer´s disease (AD), emerging evidence suggests that metabolic alterations such as type 2 diabetes (T2D) are also major contributors. Indeed, several studies have described a close relationship between AD and T2D with clinical evidence showing that both diseases coexist. A hallmark pathological event in AD is amyloid-ß (Aß) deposition in the brain as either amyloid plaques or around leptomeningeal and cortical arterioles, thus constituting cerebral amyloid angiopathy (CAA). CAA is observed in 85-95% of autopsy cases with AD and it contributes to AD pathology by limiting perivascular drainage of Aß. METHODS: To further explore these alterations when AD and T2D coexist, we have used in vivo multiphoton microscopy to analyze over time the Aß deposition in the form of plaques and CAA in a relevant model of AD (APPswe/PS1dE9) combined with T2D (db/db). We have simultaneously assessed the effects of high-fat diet-induced prediabetes in AD mice. Since both plaques and CAA are implicated in oxidative-stress mediated vascular damage in the brain, as well as in the activation of matrix metalloproteinases (MMP), we have also analyzed oxidative stress by Amplex Red oxidation, MMP activity by DQ™ Gelatin, and vascular functionality. RESULTS: We found that prediabetes accelerates amyloid plaque and CAA deposition, suggesting that initial metabolic alterations may directly affect AD pathology. T2D significantly affects vascular pathology and CAA deposition, which is increased in AD-T2D mice, suggesting that T2D favors vascular accumulation of Aß. Moreover, T2D synergistically contributes to increase CAA mediated oxidative stress and MMP activation, affecting red blood cell velocity. CONCLUSIONS: Our data support the cross-talk between metabolic disease and Aß deposition that affects vascular integrity, ultimately contributing to AD pathology and related functional changes in the brain microvasculature.

Amyloid Beta Oligomers-Induced Ca2+ Entry Pathways: Role of Neuronal Networks, NMDA Receptors and Amyloid Channel Formation.

The molecular basis of amyloid toxicity in Alzheimer's disease (AD) remains controversial. Amyloid ß (Aß) oligomers promote Ca2+ influx, mitochondrial Ca2+ overload and apoptosis in hippocampal neurons in vivo and in vitro, but the primary Ca2+ entry pathways are unclear. We studied Ca2+ entry pathways induced by Aß oligomers in rat hippocampal and cerebellar neurons. Aß oligomers induce Ca2+ entry in neurons. Ca2+ responses to Aß oligomers are large after synaptic networking and prevented by blockers of synaptic transmission. In contrast, in neurons devoid of synaptic connections, Ca2+ responses to Aß oligomers are small and prevented only by blockers of amyloid channels (NA7) and NMDA receptors (MK801). A combination of NA7 and MK801 nearly abolished Ca2+ responses. Non-neuronal cells bearing NMDA receptors showed Ca2+ responses to oligomers, whereas cells without NMDA receptors did not exhibit Ca2+ responses. The expression of subunits of the NMDA receptor NR1/ NR2A and NR1/NR2B in HEK293 cells lacking endogenous NMDA receptors restored Ca2+ responses to NMDA but not to Aß oligomers. We conclude that Aß oligomers promote Ca2+ entry via amyloid channels and NMDA receptors. This may recruit distant neurons intertwisted by synaptic connections, spreading excitation and recruiting further NMDA receptors and voltage-gated Ca2+ channels, leading to excitotoxicity and neuron degeneration in AD.

Bioavailability of once-daily tacrolimus formulations used in clinical practice in the management of De Novo kidney transplant recipients: the better study.

Multicenter, prospective, observational study to compare the relative bioavailability of once-daily tacrolimus formulations in de novo kidney transplant recipients. De novo kidney transplant recipients who started a tacrolimus-based regimen were included 14 days post-transplant and followed up for 6 months. Data from 218 participants were evaluated: 129 in the LCPT group (Envarsus) and 89 in the PR-Tac (Advagraf) group. Patients in the LCPT group exhibited higher relative bioavailability (Cmin /total daily dose [TDD]) vs. PR-Tac (61% increase; P < .001) with similar Cmin and 30% lower TDD levels (P < .0001). The incidence of treatment failure was 3.9% in the LCPT group and 9.0% in the PR-Tac group (P = .117). Study discontinuation rates were 6.2% in the LCPT group and 12.4% in the PR-Tac group (P = .113). Adverse events, renal function and other complications were comparable between groups. The median accumulated dose of tacrolimus in the LCPT group from day 14 to month 6 was 889 mg. Compared to PR-Tac, LCPT showed higher relative bioavailability, similar effectiveness at preventing allograft rejection, comparable effect on renal function, safety, adherence, treatment failure and premature discontinuation rates.

Mitochondria-ER contacts and glucose: the powerhouse of Alzheimer's disease?

A mechanism involving endoplasmic reticulum-mitochondria contacts noted in diabetes mellitus may explain the neurodegeneration and amyloidogenesis observed in these patients. Urolithin A, a metabolite found in the gut microbiome, is proposed as a therapeutic strategy for the treatment of the diabetes-related dementia.

Toll-like receptors in neuroinflammation, neurodegeneration, and alcohol-induced brain damage.

Toll-like receptors (TLRs) or pattern recognition receptors respond to pathogen-associated molecular patterns (PAMPs) or internal damage-associated molecular patterns (DAMPs). TLRs are integral membrane proteins with both extracellular leucine-rich and cytoplasmic domains that initiate downstream signaling through kinases by activating transcription factors like AP-1 and NF-κB, which lead to the release of various inflammatory cytokines and immune modulators. In the central nervous system, different TLRs are expressed mainly in microglia and astroglial cells, although some TLRs are also expressed in oligodendroglia and neurons. Activation of TLRs triggers signaling cascades by the host as a defense mechanism against invaders to repair damaged tissue. However, overactivation of TLRs disrupts the sustained immune homeostasis-induced production of pro-inflammatory molecules, such as cytokines, miRNAs, and inflammatory components of extracellular vesicles. These inflammatory mediators can, in turn, induce neuroinflammation, and neural tissue damage associated with many neurodegenerative diseases. This review discusses the critical role of TLRs response in Alzheimer's disease, Parkinson's disease, ischemic stroke, amyotrophic lateral sclerosis, and alcohol-induced brain damage and neurodegeneration.

In vivo brain imaging of mitochondrial Ca2+ in neurodegenerative diseases with multiphoton microscopy.

Mitochondria are involved in a large number of essential roles related to neuronal function. Ca2+ handling by mitochondria is critical for many of these functions, including energy production and cellular fate. Conversely, mitochondrial Ca2+ mishandling has been related to a variety of neurodegenerative diseases. Investigating mitochondrial Ca2+ dynamics is essential for advancing our understanding of the role of intracellular mitochondrial Ca2+ signals in physiology and pathology. Improved Ca2+ indicators, and the ability to target them to different cells and compartments, have emerged as useful tools for analysis of Ca2+ signals in living organisms. Combined with state-of-the-art techniques such as multiphoton microscopy, they allow for the study of mitochondrial Ca2+ dynamics in vivo in mouse models of the disease. Here, we provide an overview of the Ca2+ transporters/ion channels in mitochondrial membranes, and the involvement of mitochondrial Ca2+ in neurodegenerative diseases followed by a summary of the main tools available to evaluate mitochondrial Ca2+ dynamics in vivo using the aforementioned technique.

Mitochondria and Calcium in Alzheimer's Disease: From Cell Signaling to Neuronal Cell Death.

Mitochondrial dysfunction has been implicated in the pathogenesis of almost all neurological diseases, including Alzheimer's disease (AD). Historically, a primary focus in this context has been the link between mitochondrial dynamics and amyloid ß toxicity. Recent evidence suggests that dysregulation of mitochondrial calcium homeostasis is also related to tau and other risk factors in AD, although an ongoing challenge in the field is that data collected from different models or experimental settings have not always been consistent. We examine recent literature on mitochondrial dysregulation in AD, with special emphasis on mitochondrial calcium. We include data from in vitro systems, genetic animal models, and AD-derived human tissue, and discuss whether mitochondrial calcium transporters should be proposed as therapeutic candidates for the development of neuroprotective drugs against AD.

Therapeutic Strategies to Target Calcium Dysregulation in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people worldwide. Unfortunately, none of the current treatments are effective at improving cognitive function in AD patients and, therefore, there is an urgent need for the development of new therapies that target the early cause(s) of AD. Intracellular calcium (Ca2+) regulation is critical for proper cellular and neuronal function. It has been suggested that Ca2+ dyshomeostasis is an upstream factor of many neurodegenerative diseases, including AD. For this reason, chemical agents or small molecules aimed at targeting or correcting this Ca2+ dysregulation might serve as therapeutic strategies to prevent the development of AD. Moreover, neurons are not alone in exhibiting Ca2+ dyshomeostasis, since Ca2+ disruption is observed in other cell types in the brain in AD. In this review, we examine the distinct Ca2+ channels and compartments involved in the disease mechanisms that could be potential targets in AD.

Role of Toll Like Receptor 4 in Alzheimer's Disease.

Long-term evidence has confirmed the involvement of an inflammatory component in neurodegenerative disorders including Alzheimer's disease (AD). This view is supported, in part, by data suggesting that selected non-steroidal anti-inflammatory drugs (NSAIDs) provide protection. Additionally, molecular players of the innate immune system have recently been proposed to contribute to these diseases. Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system that recognize different pathogen-derived and tissue damage-related ligands. TLR4 mediated signaling has been reported to contribute to the pathogenesis of age-related neurodegenerative diseases, including AD. Although the pathophysiology of AD is not clear, soluble aggregates (oligomers) of the amyloid ß peptide (Aßo) have been proven to be key players in the pathology of AD. Among others, Aßo promote Ca2+ entry and mitochondrial Ca2+ overload leading to cell death in neurons. TLR4 has recently been found to be involved in AD but the mechanisms are unclear. Our group recently reported that lipopolysaccharide (LPS), a TLR4 receptor agonist, increases cytosolic Ca2+ concentration leading to apoptosis. Strikingly, this effect was only observed in long-term cultured primary neurons considered a model of aging neurons, but not in short-term cultured neurons resembling young neurons. These effects were significantly prevented by pharmacological blockade of TLR4 receptor signaling. Moreover, TLR4 expression in rat hippocampal neurons increased significantly in aged neurons in vitro. Therefore, molecular patterns associated with infection and/or brain cell damage may activate TLR4 and Ca2+ signaling, an effect exacerbated during neuronal aging. Here, we briefly review the data regarding the involvement of TLR4 in AD.

High mitochondrial calcium levels precede neuronal death in vivo in Alzheimer's disease.

Alzheimer's disease (AD), the most common cause of dementia, affects millions of people worldwide. Suggested mechanisms of neurotoxicity in AD include impaired calcium (Ca2+) homeostasis and mitochondrial dysfunction, both contributing to neuronal damage. Little was known about the exact mitochondrial Ca2+ homeostasis in the living brain, particularly in AD. Only now, with the development of intravital imaging techniques and transgenic mouse models of the disease, we are able to directly observe Ca2+ levels in specific regions or particular subcellular compartments of cells, such as mitochondria. Using multiphoton microscopy, a Ca2+ reporter targeted to mitochondria and a mouse model of cerebral ß amyloidosis (APP/PS1), our recent study (Nat Comms 2020, 11:2146) found elevated mitochondrial Ca2+ concentration in the transgenic mouse after plaque deposition, and after topical application of natural soluble amyloid beta (Aß) oligomers to the healthy mouse brain at concentrations similar to those found in the human brain. Elevated Ca2+ in mitochondria preceded neuronal death and could be targeted for neuroprotective therapies in AD. Here, we describe our main findings and pose new questions for future studies aimed at better understanding mitochondrial Ca2+ dyshomeostasis in AD.

Increased mitochondrial calcium levels associated with neuronal death in a mouse model of Alzheimer's disease.

Mitochondria contribute to shape intraneuronal Ca2+ signals. Excessive Ca2+ taken up by mitochondria could lead to cell death. Amyloid beta (Aß) causes cytosolic Ca2+ overload, but the effects of Aß on mitochondrial Ca2+ levels in Alzheimer's disease (AD) remain unclear. Using a ratiometric Ca2+ indicator targeted to neuronal mitochondria and intravital multiphoton microscopy, we find increased mitochondrial Ca2+ levels associated with plaque deposition and neuronal death in a transgenic mouse model of cerebral ß-amyloidosis. Naturally secreted soluble Aß applied onto the healthy brain increases Ca2+ concentration in mitochondria, which is prevented by blockage of the mitochondrial calcium uniporter. RNA-sequencing from post-mortem AD human brains shows downregulation in the expression of mitochondrial influx Ca2+ transporter genes, but upregulation in the genes related to mitochondrial Ca2+ efflux pathways, suggesting a counteracting effect to avoid Ca2+ overload. We propose lowering neuronal mitochondrial Ca2+ by inhibiting the mitochondrial Ca2+ uniporter as a novel potential therapeutic target against AD.

Remodeling of Intracellular Ca2+ Homeostasis in Rat Hippocampal Neurons Aged In Vitro.

Aging is often associated with a cognitive decline and a susceptibility to neuronal damage. It is also the most important risk factor for neurodegenerative disorders, particularly Alzheimer's disease (AD). AD is related to an excess of neurotoxic oligomers of amyloid ß peptide (Aßo); however, the molecular mechanisms are still highly controversial. Intracellular Ca2+ homeostasis plays an important role in the control of neuronal activity, including neurotransmitter release, synaptic plasticity, and memory storage, as well as neuron cell death. Recent evidence indicates that long-term cultures of rat hippocampal neurons, resembling aged neurons, undergo cell death after treatment with Aßo, whereas short-term cultures, resembling young neurons, do not. These in vitro changes are associated with the remodeling of intracellular Ca2+ homeostasis with aging, thus providing a simplistic model for investigating Ca2+ remodeling in aging. In vitro aged neurons show increased resting cytosolic Ca2+ concentration, enhanced Ca2+ store content, and Ca2+ release from the endoplasmic reticulum (ER). Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria is also enhanced. Aged neurons also show decreased store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway related to memory storage. At the molecular level, in vitro remodeling is associated with changes in the expression of Ca2+ channels resembling in vivo aging, including changes in N-methyl-D-aspartate NMDA receptor and inositol 1,4,5-trisphosphate (IP3) receptor isoforms, increased expression of the mitochondrial calcium uniporter (MCU), and decreased expression of Orai1/Stim1, the molecular players involved in SOCE. Additionally, Aßo treatment exacerbates most of the changes observed in aged neurons and enhances susceptibility to cell death. Conversely, the solely effect of Aßo in young neurons is to increase ER-mitochondria colocalization and enhance Ca2+ transfer from ER to mitochondria without inducing neuronal damage. We propose that cultured rat hippocampal neurons may be a useful model to investigate Ca2+ remodeling in aging and in age-related neurodegenerative disorders.

Vasomotion as a Driving Force for Paravascular Clearance in the Awake Mouse Brain.

Paravascular drainage of solutes, including ß-amyloid (Aß), appears to be an important process in brain health and diseases such as Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). However, the major driving force for clearance remains largely unknown. Here we used in vivo two-photon microscopy in awake head-fixed mice to assess the role of spontaneous vasomotion in paravascular clearance. Vasomotion correlated with paravascular clearance of fluorescent dextran from the interstitial fluid. Increasing the amplitude of vasomotion by means of visually evoked vascular responses resulted in increased clearance rates in the visual cortex of awake mice. Evoked vascular reactivity was impaired in mice with CAA, which corresponded to slower clearance rates. Our findings suggest that low-frequency arteriolar oscillations drive drainage of solutes. Targeting naturally occurring vasomotion in patients with CAA or AD may be a promising early therapeutic option for prevention of Aß accumulation in the brain.

In vivo detection of tau fibrils and amyloid ß aggregates with luminescent conjugated oligothiophenes and multiphoton microscopy.

The detection of amyloid beta deposits and neurofibrillary tangles, both hallmarks of Alzheimer's disease (AD), is key to understanding the mechanisms underlying these pathologies. Luminescent conjugated oligothiophenes (LCOs) enable fluorescence imaging of these protein aggregates. Using LCOs and multiphoton microscopy, individual tangles and amyloid beta deposits were labeled in vivo and imaged longitudinally in a mouse model of tauopathy and cerebral amyloidosis, respectively. Importantly, LCO HS-84, whose emission falls in the green region of the spectrum, allowed for the first time longitudinal imaging of tangle dynamics following a single intravenous injection. In addition, LCO HS-169, whose emission falls in the red region of the spectrum, successfully labeled amyloid beta deposits, allowing multiplexing with other reporters whose emission falls in the green region of the spectrum. In conclusion, this method can provide a new approach for longitudinal in vivo imaging using multiphoton microscopy of AD pathologies as well as other neurodegenerative diseases associated with protein aggregation in mouse models.

ABO-incompatible living-donor kidney transplantation: Study of 48 patients after desensitisation. / Trasplante renal de donante vivo ABO incompatible. Estudio de 48 pacientes tras desensibilización.

BACKGROUND: ABO-incompatible living-donor kidney transplantation was regarded as an absolute contraindication. However, it has been carried out for years with good outcomes. OBJECTIVE: Our aim was to show the results obtained with this technique in our hospital. METHODS: Forty-eight patients with a mean age of 50.9±10.9 years were included. Follow-up was 44.6±30.9 months. Conditioning: rituximab 375mg/m2, tacrolimus, mycophenolate mofetil or mycophenolate sodium, prednisone, plasmapheresis/immunoadsorption and intravenous immunoglobulin. Accepted IgG and IgM titres for transplantation:<1:8. RESULTS: Pre-process IgG titre 1:124±1:140, IgM titre 1:77±1:55. After 6±3 sessions, IgG decreased to<1:8 in 47 patients and to<1:16 in one. IgM was<1:8 in all cases. Twenty-four patients (50%) had haematoma, 7 re-intervention (14.6%), 29 (60%) required transfusion. At 5 years, acute rejection had occurred in 5 cases (8.7%), CMV infection in 9 (19.7%), BK viraemia in 5 (12.4%), post-transplant diabetes in 10 (23.4%) and lymphocele in 3 (6.4%). Patient survival was 97.1% at 5 years and graft survival 95.7% at one year and 93% at 5 years. Causes of graft loss: thrombosis (n=1); mixed rejection (n=1); and death (n=2). Serum creatinine levels were 1.4±0.4mg/dl at one and 3 years and 1.3±0.3mg/dl at 5 years. Proteinuria was 0.2±0.2g/24h at one, 3 and 5 years. CONCLUSIONS: ABO-incompatible living-donor kidney transplantation after conditioning with rituximab, plasmapheresis/immunoadsorption and immunoglobulins is a valid option offering excellent outcomes. There is a low incidence of acute rejection and no increase in infectious complications. An increased tendency for postoperative bleeding was found.

Frequency-dependent exacerbation of Alzheimer's disease neuropathophysiology.

Neuronal activity patterns are disrupted in neurodegenerative disorders, including Alzheimer's disease (AD). One example is disruption of corticothalamic slow oscillations responsible for sleep-dependent memory consolidation. Slow waves are periodic oscillations in neuronal activity occurring at frequencies of <1 Hz. The power, but not the frequency of slow oscillations is altered in a mouse model of AD. Optogenetic rescue of slow oscillations by increasing activity in cortical pyramidal neurons at the frequency of slow waves restores slow wave power, halts deposition of amyloid plaques and prevents neuronal calcium dysregulation. Here we determined whether driving this circuit at an increased rate would exacerbate the amyloid-dependent calcium dyshomeostasis in transgenic mice. Doubling the frequency of slow waves for one month with optogenetics resulted in increased amyloid beta - dependent disruptions in neuronal calcium homeostasis and loss of synaptic spines. Therefore, while restoration of physiological circuit dynamics is sufficient to abrogate the progression of Alzheimer's disease pathology and should be considered an avenue for clinical treatment of AD patients with sleep disorders, pathophysiological stimulation of neuronal circuits leads to activity - dependent acceleration of amyloid production, aggregation and downstream neuronal dysfunction.