Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy.

Microglial phagocytosis of apoptotic debris prevents buildup damage of neighbor neurons and inflammatory responses. Whereas microglia are very competent phagocytes under physiological conditions, we report their dysfunction in mouse and preclinical monkey models of stroke (macaques and marmosets) by transient occlusion of the medial cerebral artery (tMCAo). By analyzing recently published bulk and single cell RNA sequencing databases, we show that the phagocytosis dysfunction was not explained by transcriptional changes. In contrast, we demonstrate that the impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrient deprivation (OND), which led to reduced process motility, lysosomal exhaustion, and the induction of a protective macroautophagy/autophagy response in microglia. Basal autophagy, in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using pharmacological and transgenic approaches. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro, where it even had a detrimental effect on microglia, suggesting that modulating microglial autophagy to optimal levels may be a hard to achieve goal. Nonetheless, our results show that pharmacological interventions, acting directly on microglia or indirectly on the brain environment, have the potential to recover phagocytosis efficiency in the diseased brain. We propose that phagocytosis is a therapeutic target yet to be explored in stroke and other brain disorders and provide evidence that it can be modulated in vivo using rapamycin.Abbreviations: AIF1/IBA1: allograft inflammatory factor 1; AMBRA1: autophagy/beclin 1 regulator 1; ATG4B: autophagy related 4B, cysteine peptidase; ATP: adenosine triphosphate; BECN1: beclin 1, autophagy related; CASP3: caspase 3; CBF: cerebral blood flow; CCA: common carotid artery; CCR2: chemokine (C-C motif) receptor 2; CIR: cranial irradiation; Csf1r/v-fms: colony stimulating factor 1 receptor; CX3CR1: chemokine (C-X3-C motif) receptor 1; DAPI: 4',6-diamidino-2-phenylindole; DG: dentate gyrus; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HI: hypoxia-ischemia; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MCA: medial cerebral artery; MTOR: mechanistic target of rapamycin kinase; OND: oxygen and nutrient deprivation; Ph/A coupling: phagocytosis-apoptosis coupling; Ph capacity: phagocytic capacity; Ph index: phagocytic index; SQSTM1: sequestosome 1; RNA-Seq: RNA sequencing; TEM: transmission electron microscopy; tMCAo: transient medial cerebral artery occlusion; ULK1: unc-51 like kinase 1.

PET-MRI nanoparticles imaging of blood-brain barrier damage and modulation after stroke reperfusion.

In an acute ischaemic stroke, understanding the dynamics of blood-brain barrier injury is of particular importance for the prevention of symptomatic haemorrhagic transformation. However, the available techniques assessing blood-brain barrier permeability are not quantitative and are little used in the context of acute reperfusion therapy. Nanoparticles cross the healthy or impaired blood-brain barrier through combined passive and active processes. Imaging and quantifying their transfer rate could better characterize blood-brain barrier damage and refine the delivery of neuroprotective agents. We previously developed an original endovascular stroke model of acute ischaemic stroke treated by mechanical thrombectomy followed by positron emission tomography-magnetic resonance imaging. Cerebral capillary permeability was quantified for two molecule sizes: small clinical gadolinium Gd-DOTA (<1 nm) and AGuIX® nanoparticles (∼5 nm) used for brain theranostics. On dynamic contrast-enhanced magnetic resonance imaging, the baseline transfer constant K trans was 0.94 [0.48, 1.72] and 0.16 [0.08, 0.33] ×10-3 min-1, respectively, in the normal brain parenchyma, consistent with their respective sizes, and 1.90 [1.23, 3.95] and 2.86 [1.39, 4.52] ×10-3 min-1 in choroid plexus, confirming higher permeability than brain parenchyma. At early reperfusion, K trans for both Gd-DOTA and AGuIX® nanoparticles was significantly higher within the ischaemic area compared to the contralateral hemisphere; 2.23 [1.17, 4.13] and 0.82 [0.46, 1.87] ×10-3 min-1 for Gd-DOTA and AGuIX® nanoparticles, respectively. With AGuIX® nanoparticles, K trans also increased within the ischaemic growth areas, suggesting added value for AGuIX®. Finally, K trans was significantly lower in both the lesion and the choroid plexus in a drug-treated group (ciclosporin A, n = 7) compared to placebo (n = 5). K trans quantification with AGuIX® nanoparticles can monitor early blood-brain barrier damage and treatment effect in ischaemic stroke after reperfusion.

Novel Cell-Based Assay for Identification of LRRK2 Inhibitors Using Its Aberrant Regulation of a Pluripotency Gene.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2), such as the G2019S mutation, are the most common cause of familial Parkinson's disease (PD). The G2019S mutation impairs neurite outgrowth. We hypothesized that those effects could be related to an altered expression of pluripotency genes, which may provide a readout for a screening assay based on LRRK2 function. Here, we show that the G2019S mutation mediates a sustained aberrant upregulation of the transcription factors Nanog and Oct4 that in wild-type are downregulated after differentiation. The aberrant regulation of Nanog can be concentration dependently reversed by LRRK2 tool inhibitors. Building on this knowledge, we developed an assay for the identification and assessment of compounds that inhibit the aberrant pathophysiological activity of mutant LRRK2. Furthermore, the aberrant neural pluripotency is consistent with Parkinson's pathophysiology and with the epidemiological association between the G2019S genotype and cancer risk.

SUCLG2 identified as both a determinator of CSF Aß1-42 levels and an attenuator of cognitive decline in Alzheimer's disease.

Cerebrospinal fluid amyloid-beta 1-42 (Aß1-42) and phosphorylated Tau at position 181 (pTau181) are biomarkers of Alzheimer's disease (AD). We performed an analysis and meta-analysis of genome-wide association study data on Aß1-42 and pTau181 in AD dementia patients followed by independent replication. An association was found between Aß1-42 level and a single-nucleotide polymorphism in SUCLG2 (rs62256378) (P = 2.5×10(-12)). An interaction between APOE genotype and rs62256378 was detected (P = 9.5 × 10(-5)), with the strongest effect being observed in APOE-ε4 noncarriers. Clinically, rs62256378 was associated with rate of cognitive decline in AD dementia patients (P = 3.1 × 10(-3)). Functional microglia experiments showed that SUCLG2 was involved in clearance of Aß1-42.

Common variants in the HLA-DQ region confer susceptibility to idiopathic achalasia.

Idiopathic achalasia is characterized by a failure of the lower esophageal sphincter to relax due to a loss of neurons in the myenteric plexus. This ultimately leads to massive dilatation and an irreversibly impaired megaesophagus. We performed a genetic association study in 1,068 achalasia cases and 4,242 controls and fine-mapped a strong MHC association signal by imputing classical HLA haplotypes and amino acid polymorphisms. An eight-residue insertion at position 227-234 in the cytoplasmic tail of HLA-DQß1 (encoded by HLA-DQB1*05:03 and HLA-DQB1*06:01) confers the strongest risk for achalasia (P=1.73×10(-19)). In addition, two amino acid substitutions in the extracellular domain of HLA-DQα1 at position 41 (lysine encoded by HLA-DQA1*01:03; P=5.60×10(-10)) and of HLA-DQß1 at position 45 (glutamic acid encoded by HLA-DQB1*03:01 and HLA-DQB1*03:04; P=1.20×10(-9)) independently confer achalasia risk. Our study implies that immune-mediated processes are involved in the pathophysiology of achalasia.

PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Mutations in the ATP13A2 gene (PARK9, OMIM 610513) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome and early-onset parkinsonism. ATP13A2 is an uncharacterized protein belonging to the P(5)-type ATPase subfamily that is predicted to regulate the membrane transport of cations. The physiological function of ATP13A2 in the mammalian brain is poorly understood. Here, we demonstrate that ATP13A2 is localized to intracellular acidic vesicular compartments in cultured neurons. In the human brain, ATP13A2 is localized to pyramidal neurons within the cerebral cortex and dopaminergic neurons of the substantia nigra. ATP13A2 protein levels are increased in nigral dopaminergic and cortical pyramidal neurons of Parkinson's disease brains compared with normal control brains. ATP13A2 levels are increased in cortical neurons bearing Lewy bodies (LBs) compared with neurons without LBs. Using short hairpin RNA-mediated silencing or overexpression to explore the function of ATP13A2, we find that modulating the expression of ATP13A2 reduces the neurite outgrowth of cultured midbrain dopaminergic neurons. We also find that silencing of ATP13A2 expression in cortical neurons alters the kinetics of intracellular pH in response to cadmium exposure. Furthermore, modulation of ATP13A2 expression leads to reduced intracellular calcium levels in cortical neurons. Finally, we demonstrate that silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. Oppositely, overexpression of ATP13A2 delays cadmium-induced mitochondrial fragmentation in neurons consistent with a neuroprotective effect. Collectively, this study reveals a number of intriguing neuronal phenotypes due to the loss- or gain-of-function of ATP13A2 that support a role for this protein in regulating intracellular cation homeostasis and neuronal integrity.

The single-molecule mechanics of the latent TGF-ß1 complex.

BACKGROUND: TGF-ß1 controls many pathophysiological processes including tissue homeostasis, fibrosis, and cancer progression. Together with its latency-associated peptide (LAP), TGF-ß1 binds to the latent TGF-ß1-binding protein-1 (LTBP-1), which is part of the extracellular matrix (ECM). Transmission of cell force via integrins is one major mechanism to activate latent TGF-ß1 from ECM stores. Latent TGF-ß1 mechanical activation is more efficient with higher cell forces and ECM stiffening. However, little is known about the molecular events involved in this mechanical activation mechanism. RESULTS: By using single-molecule force spectroscopy and magnetic microbeads, we analyzed how forces exerted on the LAP lead to conformational changes in the latent complex that can ultimately result in TGF-ß1 release. We demonstrate the unfolding of two LAP key domains for mechanical TGF-ß1 activation: the α1 helix and the latency lasso, which together have been referred to as the "straitjacket" that keeps TGF-ß1 associated with LAP. The simultaneous unfolding of both domains, leading to full opening of the straitjacket at a force of ~40 pN, was achieved only when TGF-ß1 was bound to the LTBP-1 in the ECM. CONCLUSIONS: Our results directly demonstrate opening of the TGF-ß1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-ß1 to LTBP-1 is mandatory. Interfering with mechanical activation of latent TGF-ß1 by reducing integrin affinity, cell contractility, and binding of latent TGF-ß1 to the ECM provides new possibilities to therapeutically modulate TGF-ß1 actions.

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.

Genetic mouse models of neurodegenerative diseases.

Neurodegenerative diseases are generally characterized by the selective degeneration of particular neuronal populations and the accumulation of abnormal or aggregated proteins within, but occasionally external to, neurons in affected brain regions. These diseases can be broadly classified as disorders of cognition and memory or movement, and both features can often coexist in a single disease. In recent years, the identification of genetic mutations that cause rare monogenic familial disease has revolutionized our understanding of the molecular basis of neurodegenerative disease and has provided new targets for the development of disease-modifying therapies. An essential part of this process has been the development of genetic animal models that accurately recapitulate the essential features of each disease, with particular emphasis on the use of mouse models. Such mouse models have provided unique insight into the molecular mechanism(s) through which genetic mutations precipitate neurodegeneration and produce associated clinical and pathological phenotypes. In this review, we provide an overview of the current status, uses and limitations of genetic mouse models for understanding major neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's disease and amyotrophic lateral sclerosis.

Cholecystokinin facilitates glutamate release by increasing the number of readily releasable vesicles and releasing probability.

Cholecystokinin (CCK), a neuropeptide originally discovered in the gastrointestinal tract, is abundantly distributed in the mammalian brains including the hippocampus. Whereas CCK has been shown to increase glutamate concentration in the perfusate of hippocampal slices and in purified rat hippocampal synaptosomes, the cellular and molecular mechanisms whereby CCK modulates glutamatergic function remain unexplored. Here, we examined the effects of CCK on glutamatergic transmission in the hippocampus using whole-cell recordings from hippocampal slices. Application of CCK increased AMPA receptor-mediated EPSCs at perforant path-dentate gyrus granule cell, CA3-CA3 and Schaffer collateral-CA1 synapses without effects at mossy fiber-CA3 synapses. CCK-induced increases in AMPA EPSCs were mediated by CCK-2 receptors and were not modulated developmentally and transcriptionally. CCK reduced the coefficient of variation and paired-pulse ratio of AMPA EPSCs suggesting that CCK facilitates presynaptic glutamate release. CCK increased the release probability and the number of readily releasable vesicles with no effects on the rate of recovery from vesicle depletion. CCK-mediated increases in glutamate release required the functions of phospholipase C, intracellular Ca(2+) release and protein kinase Cgamma. CCK released endogenously from hippocampal interneurons facilitated glutamatergic transmission. Our results provide a cellular and molecular mechanism to explain the roles of CCK in the brain.

Mitochondrial alterations in Parkinson's disease: new clues.

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). In particular, complex I impairment and subsequent oxidative stress have been widely demonstrated in experimental models of PD and in post-mortem PD samples. A recent wave of new studies is providing novel clues to the potential involvement of mitochondria in PD. In particular, (i) mitochondria-dependent programmed cell death pathways have been shown to be critical to PD-related dopaminergic neurodegeneration, (ii) many disease-causing proteins associated with familial forms of PD have been demonstrated to interact either directly or indirectly with mitochondria, (iii) aging-related mitochondrial changes, such as alterations in mitochondrial DNA, are increasingly being associated with PD, and (iv) anomalies in mitochondrial dynamics and intra-neuronal distribution are emerging as critical participants in the pathogenesis of PD. These new findings are revitalizing the field and reinforcing the potential role of mitochondria in the pathogenesis of PD. Whether a primary or secondary event, or part of a multi-factorial pathogenic process, mitochondrial dysfunction remains at the forefront of PD research and holds the promise as a potential molecular target for the development of new therapeutic strategies for this devastating, currently incurable, disease.

Human immunodeficiency virus type-1 protein Tat induces tumor necrosis factor-alpha-mediated neurotoxicity.

HIV-1 infection causes, with increasing prevalence, neurological disorders characterized in part by neuronal cell death. The HIV-1 protein Tat has been shown to be directly and indirectly neurotoxic. Here, we tested the hypothesis that a non-neurotoxic epitope of Tat can, through actions on immune cells, increase neuronal cell death. Tat(1-72) and a mutant Tat(1-72) lacking the neurotoxic epitope (Tat(Delta31-61)) concentration-dependently and markedly increased TNF-alpha production in macrophage-like differentiated human U937 and THP-1 cells, in mouse peritoneal macrophages and in mouse brain microglia. Tat(1-72) was but Tat(Delta31-61) was not neurotoxic when applied directly to neurons. Supernatants from U937 cells treated with either Tat(1-72) or Tat(Delta31-61) were neurotoxic and their immunoneutralization with an anti-TNF-alpha antibody decreased Tat(1-72)- and Tat(Delta31-61)-induced neurotoxicity. Together, these results demonstrate that the neurotoxic epitope of Tat(1-72) is different from the epitope that is indirectly neurotoxic following production of TNF-alpha from immune cells, and suggest that therapeutic interventions against TNF-alpha might be beneficial against HIV-1 associated neurological disorders.

Similar calcification process in acute and chronic human brain pathologies.

Cellular microcalcification observed in a diversity of human pathologies, such as vascular dementia, Alzheimer's disease, Parkinson's disease, astrogliomas, and posttraumatic epilepsy, also develops in rodent experimental models of central nervous system (CNS) neurodegeneration. Central to the neurodegenerative process is the inability of neurons to regulate intracellular calcium levels properly, and this is extensible to fine regulation of the CNS. This study provides evidence of a common pattern of brain calcification taking place in several human pathologies, and in the rat with glutamate-derived CNS lesions, regarding the chemical composition, physical characteristics, and histological environment of the precipitates. Furthermore, a common physical mechanism of deposit formation through nucleation, lineal growth, and aggregation is presented, under the modulation of protein deposition and elemental composition factors. Insofar as calcium precipitation reduces activity signals at no energy expense, the presence in human and rodent cerebral brain lesions of a common pattern of calcification may reflect an imbalance between cellular signals of activity and energy availability for its execution. If this is true, this new step of calcium homeostasis can be viewed as a general cellular adaptative mechanism to reduce further brain damage.

Purine level regulation during energy depletion associated with graded excitatory stimulation in brain.

OBJECTIVES: The formation and release of adenosine following graded excitatory stimulation of the brain may serve important physiological functions such as sleep regulation, as well as an early resistance mechanism against excitotoxicity. However, adenosine at high levels may reflect merely the results of obstructed energy metabolism. METHODS: We examined the extent to which levels of adenosine and adenylate energy charge are affected in vivo by graded excitatory stimulations of brain using unilateral intrastriatal injections of glutamatergic agents and head-focused high energy microwaving for accurate and precise measures of purines. RESULTS: Our results confirmed that adenosine levels rise when adenylate energy charge decreases and showed that these increases occurred in three distinct phases with the rate of adenosine formation in each phase increasing as tissue adenylate energy charge was further depleted. In addition, we observed that, in most cases, the effects of focal excitatory stimulation on changes in tissue purine levels were restricted spatially within the immediate vicinity of the injection site; however, when strongly depolarizing stimuli were used, changes in purine levels could be observed in adjacent and, occasionally, even in contralateral brain regions. DISCUSSION: These results provide new insight into purine regulation that occurs under physiologically relevant conditions, such as sleep and during the early stages of brain insults that induce excitotoxicity.

Calcium precipitation in acute and chronic brain diseases.

In rat brain, calcification associated with excitotoxicity has been proposed to play a protective role, whereas in human brain, nonartherosclerotic calcification is present in several pathological conditions without any clear significance. To determine if calcification can be viewed as a protective step of calcium homeostasis during chronic and acute neuronal suffering, cerebral cortex and hippocampus of patients with Alzheimer's disease, vascular dementia and neonatal hypoxia-ischemia were investigated. To investigate the human specificity, these two areas were also studied in dogs with established cognitive deficits. In all groups, calcium precipitates were observed in the cerebral parenchyma associated with neuronal damage. The cerebral cortex presented a higher degree of calcification than the hippocampus. The neonatal hypoxia-ischemia group was characterised by a higher degree of calcification, whereas the groups with lowest calcification were the Alzheimer's patients and dogs. As shown by X-ray microanalysis, in the precipitates, calcium is mainly associated with phosphorus in a form that resembles hydroxyapatites. Thus, intracellular calcium concentration associated with neuronal suffering may reduce the energy extrusion. We propose that, to help overcome excitotoxicity, calcium precipitation acts in CNS of vertebrates as a new compartment of the calcium homeostasis in which free cytoplasmic calcium ions are inactivated by phosphate ones.