Absence Of Neuropathology Research Articles

APOE genotype modifies microglial immunometabolism

AbstractBackgroundMetabolic dysfunction and neuroinflammation characterize Alzheimer’s disease (AD), but it is unclear if these two facets of the disease are linked. The E4 allele of Apolipoprotein E (APOE) is the strongest genetic risk factor for late‐onset AD and is associated with increased neuroinflammation. Recent data from our lab and others show that E4 is also associated with increased aerobic glycolysis. These two findings may be intrinsically linked through the concept of ‘immunometabolism’ ‐ an emerging paradigm that implicates increased glycolysis as a core requirement during pro‐inflammatory activation, whereas increased oxidative phosphorylation is required for anti‐inflammatory responses.MethodPrimary microglia from mice expressing human APOE isoforms were stimulated with a combination of interferon‐γ and tumor necrosis factor‐α, two pro‐inflammatory cytokines implicated in AD. Metabolic responses were measured using the Agilent Seahorse platform and targeted metabolomics. In addition, we performed single‐cell RNA sequencing on brains from APOE targeted replacement mice at 3‐, 10‐, and 18 months of age. APOE targeted replacement mice were also crossed to the 5x Familial Alzheimer’s Disease model and brains were analyzed by spatial transcriptomics.ResultSeahorse revealed increased glycolysis and decreased mitochondrial respiration in E4 microglia. Targeted metabolomics revealed increased lactate and succinate in E4 microglia, metabolites known to accumulate in pro‐inflammatory macrophages. Aged E4 brains were found to harbor a metabolically distinct cluster of microglia that expressed a signature similar to the established ‘disease associated microglia’ (DAM) phenotype, even in the absence of neuropathology. SCENIC regulon analysis linked this cluster to the transcription network of HIF1a. Ongoing experiments targeting the HIF1a pathway seek to correct the E4‐associated increased aerobic glycolysis and excess pro‐inflammatory cytokine secretion.ConclusionOur findings reveal that inflammatory stimulation, age, and amyloid each induce a distinct metabolic response in E4 microglia. The accumulation of lactate and succinate and increased glycolysis in E4 microglia indicate altered metabolic preference conducive to pro‐inflammatory activation, whereas the decreased mitochondrial respiration may preclude effective anti‐inflammatory responses. We propose that this altered metabolism skews E4 microglia towards a phenotype that favors chronic neuroinflammation and neurodegeneration. Thus, reprogramming metabolism in E4 microglia may provide a novel therapeutic avenue for the treatment of AD.

APOE genotype modifies microglial immunometabolism

AbstractBackgroundMetabolic dysfunction and neuroinflammation characterize Alzheimer’s disease (AD), but it is unclear if these two facets of the disease are linked. The E4 allele of Apolipoprotein E (APOE) is the strongest genetic risk factor for late‐onset AD and is associated with increased neuroinflammation. Recent data from our lab and others show that E4 is also associated with increased aerobic glycolysis. These two findings may be intrinsically linked through the concept of ‘immunometabolism’ ‐ an emerging paradigm that implicates increased glycolysis as a core requirement during pro‐inflammatory activation, whereas increased oxidative phosphorylation is required for anti‐inflammatory responses.MethodPrimary microglia were isolated from targeted replacement mice expressing human APOE isoforms and stimulated with pro‐inflammatory (lipopolysaccharide (LPS); IFNγ + TNFα) or anti‐inflammatory (IL‐10; IL‐4 + IL‐13) cytokines. Metabolic responses were measured using the Agilent Seahorse platform and targeted metabolomics. We also performed single‐cell RNA sequencing on brains from APOE targeted replacement mice at 3‐, 10‐, and 18 months of age +/‐ LPS.ResultSeahorse revealed increased glycolysis and decreased mitochondrial respiration in E4 microglia. Targeted metabolomics revealed increased lactate and succinate in E4 microglia, metabolites known to accumulate in pro‐inflammatory macrophages. Aged E4 brains were uniquely found to harbor a metabolically distinct subcluster of microglia that expressed a signature similar to the established ‘disease associated microglia’ phenotype, even in the absence of neuropathology. SCENIC regulon analysis linked this cluster to the transcription network of HIF1a, and increased expression of HIF1a was validated in E4 microglia. In LPS‐treated animals, two distinct microglia subclusters emerged in E4 brains that were defined by altered expression of genes relating to oxidative phosphorylation and mitochondrial function.ConclusionOur findings reveal that age and LPS treatment induce a distinct metabolic response in E4 microglia. The accumulation of lactate and succinate and increased glycolysis in E4 microglia indicate altered metabolic preference conducive to pro‐inflammatory activation, whereas the decreased mitochondrial respiration may preclude effective anti‐inflammatory responses. We propose that this altered metabolism skews E4 microglia towards a phenotype that favors chronic neuroinflammation and neurodegeneration. Thus, reprogramming metabolism in E4 microglia may provide a novel therapeutic avenue for the treatment of AD.

Effects of Combined and General Anesthesia on Cognitive Functions for Patients Undergoing Cardiac Surgery Under CPB.

Patients may experience a variety of neurological complications after heart surgery. The most common complication observed in clinical practice is delayed neurocognitive recovery (dNCR). The role of the anesthesiologist is very important, as the risk of dNCR may be reduced, depending on the anesthesia tactic chosen. Although the possibility that neuropsychological complications are less common in patients undergoing combined anesthesia (general + epidural) than in patients undergoing general anesthesia is not yet confirmed, the results are being discussed. The aim of this study was to determine impact of combined anesthesia (general + epidural) on cognitive functions of patients after cardiac surgery. The prospective, case-controlled study included 80 patients undergoing cardiac surgery from 2015 to 2017 at the Department of Cardiothoracic and Vascular Surgery in the Hospital of Lithuanian University of Health Sciences Kauno Klinikos. After approval from the local bioethics center, informed consent was obtained from all study participants. Inclusion criteria were age 51 to 80 years, elective cardiac surgery, left ventricular ejection fraction > 35%, anamnesis of not using agents affecting the central nervous system, absence of neuropathology, and sufficient renal function. Exclusion criteria were patients suffering from diseases causing cognitive function or using agents affecting the central nervous system, emergency or re-surgery, carotid artery atherosclerosis with artery diameter 50 or more percent reduction, and a patient's disagreement. MMSE test and 6-CIT test were used for a cognitive function assessment, Trail making test and WAIS Digital Symbol Substitution test were used for psychomotor function assessment. All tests were used a day before surgery and seven days after surgery. According to the planned anesthesia, patients were assigned into two groups: 1 - combined general + epidural anesthesia and 2 - general anesthesia. Standardized protocol of anesthesia was followed for all patients. Preoperative patients and surgery factors, preoperative and postoperative neuropsychological test results were recorded. Eighty patients were enrolled in the study. Both groups did not differ in demographic, perioperative values, and baseline (preoperative) test results. Postoperative (7th day) WAIS (P = .042) and 6-item cognitive impairment (P = .016) test results were statistically different when comparing the GA and CA groups. Comparing preoperative and postoperative test results, there was a significant decline in the WAIS test score in the GA group (P = .013).

Absence of Neuropathology With Prolonged Isoflurane Sedation in Healthy Adult Rats.

The use of isoflurane sedation for prolonged periods in the critical care environment is increasing. However, isoflurane-mediated neurotoxicity has been widely reported. The goal of the present study was to determine whether long-term exposure to low-dose isoflurane in mechanically ventilated rodents is associated with evidence of neurodegeneration or neuroinflammation. Adult female Sprague-Dawley rats were used in this study. Experimental animals (n=11) were induced with 1.5% isoflurane, intubated, and given a neuromuscular blockade with α-cobratoxin. EEG electrodes were surgically implanted, subcutaneous precordial EKG Ag wire electrodes, and bladder, femoral artery, and femoral vein cannulas permanently placed. After these procedures, the isoflurane concentration was reduced to 0.5% and, in conjunction with the neuromuscular blockade, continued for 7 days. Arterial blood gases and chemistry were measured at 3 time points and core body temperature servoregulated and maintenance IV fluids were given during the 7 days. Experimental animals and untreated controls (n=9) were euthanized on day 7. Immunohistochemical and cytochemical assays did not detect evidence of microgliosis, astrocytosis, neuronal apoptosis or necrosis, amyloidosis, or phosphorylated-tau accumulation. Blood glucose levels were significantly reduced on days 3/4 and 6/7 and partial pressure of oxygen was significantly reduced, but still within the normal range, on day 6/7. All other blood measurements were unchanged. No neuropathologic changes consistent with neurotoxicity were detected in the brain after 1 week of continuous exposure to 0.5% isoflurane in healthy rats. These data suggest that even long exposures to low concentrations of isoflurane have no overt consequences on neuropathology.

Increased number of microglia in the brain of severe combined immunodeficient (SCID) mice

To assess the in vivo influence of the systemic immune system upon microglia, six defined brain regions of adult SCID mice (n = 10) lacking functional T- and B-lymphocytes have been analyzed by NDPase histochemistry, morphometry and immunohistochemistry. Despite absence of neuropathology and lack of microglial activation, microglial numerical density was significantly increased in SCID mice. Elevation was most marked in the cerebellar granular layer (by 32.6%; 95% confidence interval: 9.9-58.7%), followed by the fimbria hippocampi and the molecular layer of hippocampal CA1/CA3 region. These data need to be taken into account when using SCID mice as a model for microglial reaction in immunodeficient mice.

Reversal of learning deficits in a Tsc2+/− mouse model of tuberous sclerosis

Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) are commonly affected with specific neuropsychological problems, including long-term and working memory deficits. Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene (Tsc2(+/-) mice) show deficits in learning and memory. Cognitive deficits in Tsc2(+/-) mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved. We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder.

Recurrent spontaneous ”neuroleptic malignant syndrome” in the absence of neuroleptic medication in probable dementia with Lewy bodies

Sirs: Dementia with Lewy bodies (DLB) is a frequent neurodegenerative dementing disorder [1]. In addition to cognitive impairments, features of DLB include psychiatric symptoms (e. g., visual hallucinations), parkinsonian features, fluctuations of cognition and consciousness, and increased sensitivity to neuroleptic drugs [2]. The most severe form of the neuroleptic sensitivity reaction is neuroleptic malignant syndrome (NMS) [3]. The incidence of NMS is substantially higher in DLB patients than in other dementias [4]. This observation suggests that brain sites which are crucial to the pathogenesis of NMS represent a specific component of the degenerative process in DLB. This hypothesis would be strongly supported if NMS occurred in DLB spontaneously. A 75-year-old woman with a three-year history of dementia including episodes of disorientation and confusion was admitted because of impairment of consciousness progressing to coma within 24 hours. Over the past 2–3 years, the patient had developed a gait disturbance (small steps, slowing of movements and stooped posture). Frequent unexplained falls were noted. On admission the comatose patient displayed marked generalized rigidity. Upon stimulation, posturing of extremities was noted. Intermittently, myoclonic jerks were observed. Plantar responses were upgoing bilaterally. Temperature was 40.2 °C, blood pressure 210/120 mmHg, pulse rate 130/min, and respiratory rate was 27/min. Excessive sweating was noted. The remainder of the medical examination was unremarkable. Two similar episodes had led to admissions in other institutions one month and one year earlier with insidiously developing “stupor”, hyperthermia, increased muscle tone, tachycardia, and markedly elevated serum levels of CK. These episodes had been fully reversible. Extensive enquiries did not reveal intake of neuroleptics or dopaminergic drugs or any other drug known to be related to NMS. CK on admission was elevated (118 U/l) and rose to 606 U/l on the following day. Infection, metabolic causes, epileptic seizures, intoxication, or cerebral ischemia were ruled out. Beta-amyloid 1–42 was decreased, while τ-protein was increased. Brain imaging studies revealed marked generalized atrophy and minimal signs of microangiopathy. Fluor-desoxyglucose positron emission tomography (FDG-PET) studies showed reduction of tracer uptake in the parieto-occipital region bilaterally and in the left basal ganglia, on top of a diffuse cerebral hypometabolism (Fig. 1a). Single photon emission computed tomography (SPECT) using I-[123]-Beta carbomethoxy-iodophenyltropane (CIT) and I-[123]-Iodobenzamid (IBZM) suggested reduced density of presynaptic dopamine carriers at the level of the basal ganglia bilaterally, and reduction of postsynaptic D2-dopamine receptors in the left basal ganglia, respectively (Fig. 1b, c). Following improvement over 14 days the patient suffered a spontaneous relapse with stupor, gaze deviation, increased muscle tone, myoclonic jerking and hyperthermia. From this, she recovered incompletely. This appears to be the first report of the spontaneous occurrence of an NMS-like condition in severe degenerative dementia. Although a final diagnosis cannot be made in the absence of neuropathology, findings strongly point to DLB as the underlying dementing disorder. Our patient exhibited a cognitive decline including episodes of confusion, and motor features of parkinsonism suggesting a diagnosis of “probable DLB” [2]. This diagnosis is further supported [2] by the symptoms “repeated falls” and “transient losses of consciousness”. In addition, FDG-PET showed parieto-occipital cerebral hypometabolism, in agreement with the specific pattern of DLB-patients found in imaging studies [5, 6] and at variance from the pattern associated with Alzheimer’s disease [5, 6]. Furthermore, PET imaging, IBZM-SPECTand 123-I-βCIT-SPECT-findings suggested basal ganglia hypometabolism and basal ganglia pre-and postsynaptic impairment of dopaminergic neurotransmission consistent with findings obtained in DLB-patients, but not in Alzheimer’s disease [7]. Decreased β-amyloid-levels [8] and increased τ-protein levels [9] are consistent with DLB. The syndrome of hyperthermia, rigidity, elevated CK and severely impaired consciousness with optional tachycardia, tachypnea, hypertension and diaphoresis has been named “neuroleptic malignant syndrome” although it may occur also in the absence of expoLETTER TO THE EDITORS

Human apoE4-targeted replacement mice display synaptic deficits in the absence of neuropathology

The human APOE*4 allele is associated with an early age of onset and increased risk of Alzheimer's disease (AD). Long before the onset of AD, cognitive deficits can be identified in APOE*4 carriers. We examined neurons in the lateral amygdala of young apolipoprotein (apo) E3 and apoE4 targeted replacement (TR) mice for changes in synaptic integrity. ApoE4 mice displayed significantly reduced excitatory synaptic transmission and dendritic arborization. Despite these changes there were no signs of gliosis, amyloid deposition or neurofibrillary tangles in these mice. To our knowledge, this is the first study to suggest that cognitive deficits in APOE*4 carriers are due to inherent defects in synaptic function that appear prior to any age-dependent markers of neuropathology.

On the cause of mental retardation in Down syndrome: extrapolation from full and segmental trisomy 16 mouse models

Down syndrome (DS, trisomy 21, Ts21) is the most common known cause of mental retardation. In vivo structural brain imaging in young DS adults, and post-mortem studies, indicate a normal brain size after correction for height, and the absence of neuropathology. Functional imaging with positron emission tomography (PET) shows normal brain glucose metabolism, but fewer significant correlations between metabolic rates in different brain regions than in controls, suggesting reduced functional connections between brain circuit elements. Cultured neurons from Ts21 fetuses and from fetuses of an animal model for DS, the trisomy 16 (Ts16) mouse, do not differ from controls with regard to passive electrical membrane properties, including resting potential and membrane resistance. On the other hand, the trisomic neurons demonstrate abnormal active electrical and biochemical properties (duration of action potential and its rates of depolarization and repolarization, altered kinetics of active Na +, Ca 2+ and K + currents, altered membrane densities of Na + and Ca 2+ channels). Another animal model, the adult segmental trisomy 16 mouse (Ts65Dn), demonstrates reduced long-term potentiation and increased long-term depression (models for learning and memory related to synaptic plasticity) in the CA1 region of the hippocampus. Evidence suggests that the abnormalities in the trisomy mouse models are related to defective signal transduction pathways involving the phosphoinositide cycle, protein kinase A and protein kinase C. The phenotypes of DS and its mouse models do not involve abnormal gene products due to mutations or deletions, but result from altered expression of genes on human chromosome 21 or mouse chromosome 16, respectively. To the extent that the defects in signal transduction and in active electrical properties, including synaptic plasticity, that are found in the Ts16 and Ts65Dn mouse models, are found in the brain of DS subjects, we postulate that mental retardation in DS results from such abnormalities. Changes in timing and synaptic interaction between neurons during development can lead to less than optimal functioning of neural circuitry and signaling then and in later life.

No changes in behaviour, nigro-striatal system neurochemistry or neuronal cell death following toxic multiple oral paraquat administration to rats.

We have examined whether the widely used herbicide, paraquat (1,1'-dimethyl-4,4'dipyridylium) may accumulate in rat brain following multiple oral dosing (5 mg paraquat ion/kg/day) for 14 days and whether this dosing regime may produce signs of neurotoxicity. This dosing regime may determine whether low dose exposure to mammals may be neurotoxic. Using [14C]paraquat to measure tissue and plasma paraquat concentrations, we observed significantly higher plasma and tissue paraquat concentrations in brain, liver, lungs and kidneys of rats which received multiple doses for 14 days, as compared to paraquat concentrations in tissues of rats which received only a single paraquat dose. Brain paraquat concentrations measured 24 h after dosing were tenfold higher in rats receiving 14 daily oral doses of paraquat, as compared to concentrations following a single oral dose. A neuropathological study of the rat brain yielded no evidence that multiple paraquat dosing resulted in neuronal cell damage. Particular attention was paid to the nigrostriatal system. The paraquat treated rats gained approximately 10% less body weight over the 15 day experimental period as compared with controls demonstrating that the dose of paraquat was toxic to the animals. Measurements of locomotor activity using open field tests or activity monitors did not reveal any statistically significant differences between control animals and those receiving paraquat. Fore- and hind-limb grip strength were not significantly different between the paraquat treated and control rats at any time point during the dosing regime, nor was there any evidence for locomotor coordination deficits in any of the animals receiving paraquat. Densities of dopamine D1 and D2, NMDA, muscarinic and benzodiazepine receptors in the cerebral cortex and striatum were not significantly different between controls and rats which had received multiple paraquat doses. Concentrations of catecholamine neurotransmitters in the striatum, hypothalamus and frontal cerebral cortex were also measured to examine whether there was evidence for catecholamine depletion in these brain regions. We did not observe any significant reductions in dopamine, noradrenaline or DOPAC concentrations in any brain region of paraquat treated rats as compared with controls. On the contrary, dopamine concentrations in the striatim were significantly elevated in paraquat treated animals following a 15 day paraquat dosing regime. We attribute these changes in catecholamine concentrations to the general toxicity of paraquat which produces a stress response. In conclusion, we could not find any evidence that multiple paraquat dosing can lead to changes in locomotor activity or grip strength. In addition, the absence of neuropathology or changes in neurochemistry in the nigrostriatal tract demonstrates that paraquat does not behave like MPP+(N-methyl-4-phenylpyridinium), the neurotoxic metabolite of MPTP.

Rapid and reversible astrocytic reaction to afferent activity blockade in chick cochlear nucleus

We describe here rapid proliferation of astrocytic processes immunoreactive for glial fibrillary acidic protein (GFAP) in the chick cochlear nucleus following blockade of action potentials in the afferent nerve. Unilateral eighth nerve activity blockade was achieved through intralabyrinthine injection of TTX. Within 1 hr of activity blockade, a 56% increase in area density of GFAP-immunoreactive processes was found in the ipsilateral cochlear nucleus as compared to the contralateral side of the brain. This increase reached 152% by 3 hr. When eighth nerve activity was allowed to recover and animals were studied 1 week after TTX injection, no difference was found in GFAP immunoreactivity between the ipsilateral and contralateral cochlear nuclei. This is the first report of a glial reaction to documented neuronal inactivity in the absence of neuropathology. These results indicate that neuronal activity may regulate the structure of astrocytic processes.