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Retina and optic nerve are sites of extra-cerebral manifestations of Alzheimer's Disease (AD). Amyloid-β (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau protein are detected in eyes from AD patients and transgenic animals in correlation with inflammation, reduction of synapses, visual deficits, loss of retinal cells and nerve fiber. However, neither the pathological relevance of other post-translational tau modifications-such as truncation with generation of toxic fragments-nor the potential neuroprotective action induced by their in vivo clearance have been investigated in the context of AD retinal degeneration. We have recently developed a monoclonal tau antibody (12A12mAb) which selectively targets the neurotoxic 20-22 kDa NH<sub>2</sub>-derived peptide generated from pathological truncation at the N-terminal domain of tau without cross-reacting with its full-length normal protein. Previous studies have shown that 12A12mAb, when intravenously (i.v.)-injected into 6-month-old Tg2576 animals, markedly improves their AD-like, behavioural and neuropathological syndrome. By taking advantage of this well-established tau-directed immunization regimen, we found that 12A12mAb administration also exerts a beneficial action on biochemical, morphological and metabolic parameters (i.e. APP/Aβ processing, tau hyperphosphorylation, neuroinflammation, synaptic proteins, microtubule stability, mitochondria-based energy production, neuronal death) associated with ocular injury in the AD phenotype. These findings prospect translational implications in the AD field by: (1) showing for the first time that cleavage of tau takes part in several pathological changes occurring in vivo in affected retinas and vitreous bodies and that its deleterious effects are successfully antagonized by administration of the specific 12A12mAb; (2) shedding further insights on the tight connections between neurosensory retina and brain, in particular following tau-based immunotherapy. In our view, the parallel response we detected in this preclinical animal model, both in the eye and in the hippocampus, following i.v. 12A12mAb injection opens novel diagnostic and therapeutic avenues for the clinical management of cerebral and extracerebral AD signs in human beings. |
In a small fraction of patients, intracranial meningiomas arise as multiple and spatially distinct masses therefore presenting a unique management challenge [ , , ]. A recently-published, (Surveillance, Epidemiology, and End Results) SEER-based study has reported that patients with multiple meningiomas (MM) have substantially reduced overall survival when compared to patients with single meningiomas [ ]. Patients may develop multiple meningiomas in sporadic or hereditary forms. Familial syndromes that are commonly associated with MM are neurofibromatosis type 2 (NF2) and familial meningiomatosis in patients with germline NF2 and SMARCB1 mutations, respectively [ , ]. While the mutational landscape of single meningiomas has been extensively studied [ – , , ], understanding of the molecular pathogenesis of sporadic MM remains incomplete. Older studies and case reports have reported molecular testing in patients with sporadic MM that have principally been focused on tumors with NF2 mutations [ , – ]. However, to our knowledge, no molecular profiling in a case series of spatially separated MM, composed of different histological subtypes, has been performed. The objective of this study is to elucidate the genetic features of sporadic MM, defined as the presence of ≥ 2 spatially separated synchronous or metachronous lesions.
This series includes 17 resected sporadic meningiomas from eight patients (seven females and one male) that were identified by a record search for patients with MM. All patients presented with synchronous, spatially separated meningiomas without evidence of tumor bridging, as reviewed on MR-imaging. The patients had no significant prior radiation exposure and the tumors did not arise in patients who met the clinical criteria for the diagnosis of familial schwannomatosis or neurofibromatosis type 2 [ ]. In addition, upon reviewing cranial and spinal MR images, no patient had other intra- or extra-cranial tumors associated with hereditary meningioma syndromes such as schwannomas or ependymomas.
Fresh frozen tumor tissue was available from all 17 meningiomas and was retrieved from the archives of the Institute for Pathology at the University Hospital Dresden upon approval of the local ethics committee. Two board-certified pathologists confirmed the pathologic diagnosis of each case. All tumors were classified according to the 2016 WHO classification of tumors of the central nervous system [ ]. The tumor DNA was purified using AllPrep DNA Universal Kit for fresh frozen tissue (Qiagen, Germantown MD) following the manufacturer’s instructions. The regions of interest were amplified using a custom designed amplikon panel according to the protocol “QIAseq Targeted DNA V3 Panel, May 2017” (QIAGEN, Hilden, Germany). The panel was custom-designed by our group and manufactured by QIAGEN. The panel covers either mutation hotspots or—where loss of function is a known mechanism of action—whole genes. The following meningioma-relevant genes are included: AKT1, ATRX, CDKN2A, KLF4, NF1, NF2, PIK3CA, PIK3R1, POLR2A, PTEN, SMARCB1, SMO, STAG2, SUFU, TP53, TRAF7 , and TERT promotor. During library preparation unique molecular barcodes and sample specific indices were incorporated according to the protocol. Indexed libraries were then quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, MA, USA) and paired end sequenced (2x200 bp) on Illumina MiSeq platform. HG19 was used as reference genome for bioinformatic analyses. The bioinformatics evaluation was performed using the Biomedical Workbench from CLC (12.0.3) using a customized analysis algorithm with following filters: coverage >/=100, allele frequency >/=5%. Notably, we performed internal NGS controls for identity check and cross contamination checks to assure the assignment of the correct samples.
The average age at presentation was 60 years (range 43–75 years) which is comparable with the age of patients with single sporadic meningiomas [ ]. Six patients (75%) underwent two surgeries within 2 years for tumor resection, whereas in two patients the meningiomas were removed at the same time (patients 1 and 7). Fourteen meningiomas were WHO grade 1 (82.3%) and the remaining three tumors were WHO grade 2. This is consistent with previous reports of the predominance of WHO grade 1 among MM [ , ].
Most importantly, the same mutation was not identified in separate tumors from the same patient, suggesting genomically distinct molecular drivers and an independent origin of these multiple lesions. All but two cases harbored TRAF7 , AKT1 , SMO or PIK3CA mutations (Fig. ). The most frequent driver mutations in our series were TRAF7 (n = 5), PIK3CA H1047R and E545G (n = 3), AKT1 E17K (n = 3), NF2 (n = 2), SMO L412F (one case) and NF1 (one case). We did not detect a known driver mutation in only one meningioma (MM #3, Site B; Table ). Interestingly, with the exception of one patient (MM #5), all tumors from the same patient were different histopathological subtypes (Table ).
Illustrative cases from three patients with seven meningiomas are shown. No separate tumors within individual patients shared driver mutations
Patients’ and tumor characteristics
R right, L left, M midline
The low frequency of NF2 mutations in our MM series stands in contrast to previous studies that included hereditary cases arising in the setting of NF2 [ , , , ]. Those studies identified a high prevalence of NF2 mutations (up to 83%) and supported a monoclonal origin for MM [ , ]. Our findings in a cohort of 17 MM arising in patients without NF2 support a model in which sporadic MM can arise independently from one another, while a subset of MM may result from somatic NF2 mosaicism [ ].
Each of the meningiomas in our study exhibited features that are commonly seen in solitary meningiomas, demonstrating strong associations between the genetic alteration, the histologic subtype and the anatomic location [ , , ]. The high frequency of known and targetable drivers of meningioma in our cohort suggests that a large fraction of MM may be candidates for study in clinical trials evaluating targeted therapies, such as the ongoing multicenter phase II study (ClinicalTrials.gov NCT02523014) that investigates the efficacy of afuresertib in AKT1 -mutant, vismodegib in SMO -mutant and the focal adhesion kinase (FAK) inhibitor GSK2256098 in NF2 -mutant meningiomas. Given the inter-tumor and intra-patient heterogeneity that we observe in the setting of MM, target lesions should be genomically characterized and not assumed to share molecular alterations with separately resected lesions.
Taken together, our molecular analysis supports the genomic divergence of sporadic MM and presumably their independent origin. Our findings have important clinical implications for this patient population and suggests molecular stratification of each meningioma lesion in patients with sporadic MM to improve the design of meningioma clinical trials and help improve patient management.
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Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB -NM pathophysiological mechanisms remain unclear, histological analyses of patients’ muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB -NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin‐deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB -NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin‐deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB -NM.
## Supplementary Information
The online version contains supplementary material available at 10.1186/s40478-022-01491-9.
## Introduction
Nemaline myopathy (NM) is among the most common non-dystrophic genetic muscle disorders, with an estimated incidence of 1 in 20,000 live births [ , ]. Clinical symptoms of NM include hypotonia, muscle weakness and fatigue [ , ]. In the severe form, neonatal death may ensue whilst milder forms range from delayed motor developmental milestones to requiring a wheelchair, or even late-onset mild muscle dysfunction in adulthood [ , ]. In all forms of NM, respiratory compromise is a risk throughout life [ , ]. NEB mutations account for more than 50% of all NM cases [ ]. These mutations result in shorter forms or haplo-insufficiency of the giant protein, nebulin, known to be an integral component of the thin filaments in skeletal muscle [ , ]. Following this, we and others have observed that with shorter forms or reduced levels of nebulin, actin filament activation is incomplete. Subsequently, myosin motors cannot bind properly to actin monomers, which depresses the force-generating capacity of muscle fibres, thus causing muscle weakness in NEB -NM [ , , , ]. Hence, we have previously targeted the force production of myosin proteins in NM utilizing a recombinant adeno-associated viral vector-related gene therapy and showed its promise in an animal model with a mutation in the NM-causing gene, ACTA1 [ ]. However, this approach failed to restore muscle function in NEB -NM mouse models (unpublished data). This suggests that, despite major advancements in our understanding of the disease, NM pathophysiology is complex and far from being fully understood and consequently the design and implementation of accurate therapeutic interventions remains challenging [ ].
Remarkably, ultrastructural and histological observations from NEB -NM patients and from relevant murine models not only shed light on the presence of nemaline rods (an important diagnostic tool for NM) but also on glycogen deposits and misshapen mitochondria with noticeable pleomorphism, concentric christae and increased subsarcolemmal crescents [ , ]. In line with these observations, muscle glycolytic pathways have been found altered [ ]. These findings indicate an under-appreciated potential change in muscle energetics and metabolism in NEB -NM as well as in other forms of NM caused by other gene mutations. Further support comes from clinical observations reporting that children and adolescents with NM are often lean despite their inability to engage in fast motor activities. Inefficient binding of the force-producing myosin molecules to actin filaments may contribute to altered energetics and metabolism in NM muscles by subtly increasing the energy (ATP) cost of contraction [ , ]. Nevertheless, other more prominent pathological ATP-consuming mechanisms are likely to occur in NEB -NM. In the present study, we initially set forth to explore this hypothesis and study the involvement of resting myosin energetics, as an underlying NM mechanism.
Myosin has multiple chemo-mechanical states [ ]. In addition to several active states, two distinctive relaxed states exist; myosin heads that are detached from actin filaments, and do not produce force, can be in either ‘super-relaxed’ or ‘disordered-relaxed’ states [ , ]. In the super-relaxed state, myosin heads interact with the thick filament backbone restricting their interaction with actin. In the disordered-relaxed state, myosin molecules are not immobilized and can weakly bind actin allowing a fast transition to the active state when actin filaments are switched on. The fraction of myosin heads in disordered-relaxed and super-relaxed conformations correlates with the rate of ATP usage, with the ATPase activity of myosin heads in the disordered-relaxed configuration being ten times higher than this in the super-relaxed state [ , ]. Thus, in the present study, we further hypothesized that in NEB -NM, the proportion of myosin molecules in the super-relaxed state is disrupted, impacting the basal ATP consumption of skeletal muscle, ultimately modifying the level of proteins involved in energy-producing pathways and contributing to the disease phenotype. To test this hypothesis, we used skeletal myofibres extracted from a wide spectrum of NM patients as well as from a muscle-specific nebulin conditional knockout mouse model (cNEB KO). We then performed a combination of biophysical assays, cell biology techniques and proteomics analyses. Interestingly, in line with our hypotheses, we found that in relaxed muscle fibres from NM patients, the myosin-stabilizing structural state is altered, with a potential causal involvement of myosin binding proteins such as regulatory light chains and myosin-binding protein C that are known to be involved in sequestering the super-relaxed state. We also observed that the increase in basal myosin ATP consumption may remodel muscle energy proteins, altogether paving the way to therapies related to myosin for NM.
## Materials and methods
### Human subjects
Muscle biopsy specimens were obtained from 26 NM patients with known mutations in either NEB, ACTA1 , TPM2 or TPM3 ; and 12 age-matched controls with no history of neuromuscular disease. Eleven additional NM patients had an extremely rare, late-onset acquired myopathy termed sporadic late-onset NM (SLONM) that is known to have similar histopathological abnormalities as genetic NM and that progresses subacutely [ ]. All tissue was consented, stored, and used in accordance with the Human Tissue Act, UK, under local ethical approval (REC 13/NE/0373). Details of all the 49 individuals are given in Additional file : Table S1. All samples were flash-frozen and stored at − 80 °C until analyzed.
### Nebulin knockout mouse model
The conditional muscle-specific nebulin knockout mouse model used in the present study has previously been published in detail [ ]. Briefly, mice were on a C57BL/6 J background. Floxed mice were bred to a MCK-Cre strain that expresses Cre recombinase under the control of the Muscle Creatine Kinase (MCK) promoter. Mice that were positive for MCK-Cre and homozygous for the floxed nebulin allele were nebulin deficient (cNeb KO). Mice with one nebulin wild-type allele and being either MCK-Cre positive or negative served as controls. All the experiments were approved by the University of Arizona Institutional Animal Care and Use Committee (09-056) and were in accordance with the United States Public Health Service’s Policy on Humane Care and Use of Laboratory Animals. At 6 months of age, six cNeb KO and six control female mice were weighed, anesthetized with isoflurane, and sacrificed by cervical dislocation. Tibialis cranialis skeletal muscles were then dissected and flash-frozen in liquid nitrogen before being stored at − 80 °C for later analysis.
### Solutions
As previously published [ ], the relaxing solution contained 4 mM Mg-ATP, 1 mM free Mg , 10 mM free Ca , 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate and KCl to adjust the ionic strength to 180 mM and pH to 7.0. Additionally, the rigor buffer for Mant-ATP chase experiments contained 120 mM K acetate, 5 mM Mg acetate, 2.5 mM K HPO , 50 mM MOPS, 2 mM DTT with a pH of 6.8. The lambda phosphatase solution (New England Biolabs) was prepared by a 100-fold dilution into the relaxing solution to yield 4 U lambda phosphatase/µl [ ]. The solution for extracting myosin regulatory light chains (RLC) contained 20 mM EDTA, 50 mM KPr, 10 mM potassium phosphate buffer with a pH of 7.1 [ ]. Finally, the solution for extracting myosin-binding protein C (MyBP-C) contained 10 mM EDTA, 31 mM Na HPO , 124 mM NaH PO , with a pH of 5.9 [ , ].
### Muscle preparation and fibre permeabilisation
Cryopreserved human and mouse muscle samples were immersed in a membrane-permeabilising solution (relaxing solution containing glycerol; 50:50 v/v) for 24 h at − 20 °C, after which they were transferred to 4 °C and bundles of approximately 50–100 muscle fibres were dissected free. These bundles were kept in the membrane-permeabilising solution at 4 °C for an additional 24 h (to allow for a proper skinning/membrane permeabilisation process). After these steps, bundles were stored in the same buffer at − 20 °C for use up to 1 week [ , ].
### Mant-ATP chase experiments
On the day of the experiments, bundles were transferred to relaxing solution and single myofibres were manually isolated. Their ends were individually clamped to half-split copper meshes designed for electron microscopy (SPI G100 2010C-XA, width, 3 mm), which had been glued to glass slides (Academy, 26 × 76 mm, thickness 1.00–1.20 mm). Cover slips were then attached to the top (using double-sided tape) to create flow chambers (Menzel-Glaser, 22 × 22 mm, thickness 0.13–0.16 mm) [ ]. Muscle fibres were mounted at a relaxed length (with their sarcomere length checked using the brightfield mode of a Zeiss Axio Scope A1 microscope, approximately at 2.20 µm). Similar to previous studies [ ], all experiments were performed at 25 °C, and each fibre was first incubated for 5 min with a rigor buffer. A solution containing the rigor buffer with 250 μM Mant-ATP was then flushed and kept in the chamber for 5 min. At the end of this step, another solution made of the rigor buffer with 4 mM unlabelled ATP was added with simultaneous acquisition of the Mant-ATP chase.
For fluorescence acquisition, a Zeiss Axio Scope A1 microscope was used with a Plan-Apochromat 20x/0.8 objective and a Zeiss AxioCam ICm 1 camera. Frames were acquired every 5 s for the first 90 s and every 10 s for the remaining time with a 20 ms acquisition/exposure time using a DAPI filter set, and images were collected for 5 min. Three regions of each individual myofibre were sampled for fluorescence decay using the ROI manager in ImageJ as previously published [ ]. The mean background fluorescence intensity was subtracted from the average of the fibre fluorescence intensity (for each image taken). Each time point was then normalized by the fluorescence intensity of the final Mant-ATP image before washout (T = 0). These data were then fit to an unconstrained double exponential decay using Graphpad Prism 9.0: where P1 is the amplitude of the initial rapid decay approximating the disordered-relaxed state with T1 as the time constant for this decay. P2 is the slower second decay approximating the proportion of myosin heads in the super-relaxed state with its associated time constant T2 [ ].
### Immunofluorescence staining and imaging
To avoid any potential misinterpretation due to the type of myosin heavy chain, for the human Mant-ATP chase experiments, we assessed the sub-type using immunofluorescence staining as previously described [ ]. Briefly, flow-chamber mounted myofibres were stained with an anti-β-cardiac/skeletal slow myosin heavy antibody (IgG1, A4.951, sc-53090 from Santa Cruz Biotechnology, dilution: 1:50) and an anti-slow myosin binding protein C antibody (IgG, SAB3501005 from Sigma, dilution: 1:200). Myofibres were then washed in PBS/0.025% Tween-20 and incubated with secondary antibodies: goat anti-mouse IgG1 Alexa 555 and goat anti-rabbit IgG Alexa 488 (from ThermoScientific, dilution 1:1000), respectively, in a blocking buffer. After washing, muscle fibres were mounted in Fluoromount. To identify the type of fibres, images were acquired using a confocal microscope (Zeiss Axiovert 200, 63 × oil objective) equipped with a CARV II confocal imager (BD Biosciences) [ , ]. To obtain myosin filament length and myosin-binding protein C (MyBP-C) localisation measurements, mounted muscle fibres were acquired with a 100 × oil objective and an instant Structured Illumination Microscope (iSIM) system. To improve contrast and resolution (by two-fold compared to confocal microscopy), distributed deconvolution (DDecon) was then applied from the acquired images with a specific plugin for ImageJ (National Institutes of Health, Bethesda, MD) [ ]. Note that DDecon is a super-resolution light microscopy technique that addresses light scattering, differences in refractive index, glare, and background noise. It also allows the computation of filament lengths with a precision of 10–20 nm [ , ]. All line scans were background corrected. Distances and lengths were finally calculated by converting pixel sizes into µm using the scale for each image [ , ].
### Western blotting
Lysates of the flash-frozen human muscle biopsy specimens from three control subjects and three NEB -NM patients were prepared via hand-homogenization in a modified NP-40 lysis buffer (10 mM NaH PO , pH 7.2, 2 mM EDTA, 10 mM NaN , 120 mM NaCl, 0.5% deoxycholate, 1% NP-40) supplemented with complete protease inhibitor (Roche, Indianapolis, IN) and Halt phosphatase inhibitor (Thermo Scientific, Waltham, MA) cocktails. NuPage LDS sample buffer and reducing agent (Invitrogen, Waltham, MA) were added to 30 μg of protein lysate, boiled at 95 °C for 5 min, and fractionated by 4–12% SDS-PAGE. Protein was transferred to nitrocellulose membrane, blocked with 5% milk (RPI, Mt Prospect, IL) in TBST, and probed with the appropriate primary antibody: anti-slow myosin binding protein C (sMyBP-C, SAB3501005, Sigma-Aldrich, St. Louis, MO), anti-GAPDH (G7895, Sigma-Aldrich, St. Louis, MO), and custom phospho-sMyBP-C specific antibodies against mSer-59/hSer59 and mThr-84/hSer-82 as described previously [ ]. Blots were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA) and ECL substrate (Thermo Scientific, Waltham, MA). Densitometry was performed with ImageJ software. Total sMyBP-C blots produced a non-specific band of higher protein mass. Only the bottom specific band of the correct size was used for quantification. Relative sMyBP-C phosphorylation was calculated based on the sample’s total level of sMyBP-C following normalization to GAPDH loading control (Additional file : Fig. S1A–C).
### Enzymatic isolation and culture of intact mouse single muscle fibres
All animal procedures associated with enzymatic isolation of single muscle fibres were carried out at King’s College London in accordance with the UK Home Office regulations and in compliance with the European Community Directive published in 1989 (86/609/EEC). Two adult mature C57BL/6J mice were euthanized using cervical dislocation at 8 weeks of age. As previously published [ ], Extensor digitorum longus (EDL) skeletal muscles were dissected, leaving tendons intact at both the proximal and distal ends. Subsequently, muscles were digested in 2 mg/mL collagenase I (Sigma Aldrich) in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) for 105 min. Single fibres were released via trituration with a wide-bore glass pipette and hypo-contracted fibres and debris were removed following serial washes. Freshly isolated fibres were moved into six well plates; after 1 h they were dosed with either 100 µM of piperine or dimethyl sulfoxide (DMSO), which served as control, which was left for 3 days at 37 °C and 5% CO . These fibres were then subjected to LC–MS/MS.
### LC–MS/MS identification and quantitative analysis of protein abundance
As previously published [ , ], five samples for each experimental group were prepared. Each sample consisted of five single muscle fibres into a single centrifuge tube containing 30 μL Tris-Triton lysis buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, protease inhibitor cocktail III (1:100), phosphatase inhibitor cocktail mix (1:100) at an unknown protein concentration). Sample volume was reduced by half in a SpeedVac (ThermoFisher Scientific) and subsequently mixed in a 1:1 ratio with Laemmli buffer (2 × conc.), vortexed and boiled at 96 °C for 10 min. To stack the protein complement and remove chemical interference from the lysis buffer, samples were centrifuged at 14,000 rpm for 3 min prior to loading in 10% BisTris gels (Gel 1—ThermoFisher Scientific #19072670-1957; Gel 2—#19072670-1965; Gel 3—#19072670-1966; Gel 4—#19072670-1977). Gels were then stained overnight with Imperial protein stain (ThermoFisher Scientific #24615). In-gel reduction, alkylation and digestion with trypsin was performed prior to subsequent isobaric mass tag labelling. Each sample was treated individually with labels (TMT10plex) added at a 1:1 ratio [ ].
For analysis by LC–MS/MS, TMT labelled peptide samples were resuspended in 60 μL of resuspension buffer (2% ACN in 0.05% FA) with 10 μL sample injected in triplicate (30 μL total volume). Chromatographic separation was performed using an Ultimate 3000 NanoLC system (ThermoFisher Scientific). Peptides were resolved by reversed phase chromatography on a 75 μm × 50 cm C18 column using a three-step gradient of water in 0.1% formic acid and 80% acetonitrile in 0.1% formic acid. The gradient was delivered to elute the peptides at a flow rate of 250 nl/min over 250 min. The eluate was ionised by electrospray ionisation using an Orbitrap Fusion Lumos (ThermoFisher Scientific) operating under Xcalibur v4.1.
#### Database searching
Raw mass spectrometry data from the triplicate injection were processed into peak list files using Proteome Discoverer (ThermoScientific; v2.2) (PD 2.2). The data was processed and searched using the Mascot search algorithm (v2.6.0) and the Sequest search algorithm [ ] against the Uniprot Mouse Taxonomy database (36,483 entries). Within the consensus processing module, the reporter ion intensity values (absolute area under the peak) for each peptide spectral match are grouped with peptides and calculated at the protein level identification as a grouped abundance. All grouped abundances at protein level are normalised using total peptide amount which has previously been corrected based on the highest peptide abundance present in one channel, thus all channels have the same total abundance.
#### Bioinformatics and data visualisations
Following processing with Proteome Discoverer, the resultant file was exported into Perseus (v1.6.3) for qualitative and quantitative data analysis. Metascape [ ] was utilised for gene ontological (GO) analysis, which was subsequently visualised using cytoscape [ ]. DAVID bioinformatic database was used for ligand binding analysis [ ]. Further data visualisation utilised Biovinci v3.0.9 and Graphpad Prism v9.
#### Statistical analysis
Multiple myofibres were studied for each subject. Hence, as previously published [ ], we used mixed linear models to statistically analyze the data. These models assumed that each subject had its own mean measurement (with a normal distribution between subjects) and that each measurement within a subject was also normally distributed around this mean. The p values tested the hypotheses that there were differences in these mean measurements between groups. Data are then presented as means ± standard deviations. Graphs were prepared and analyzed in Graphpad Prism v9. Statistical significance was set to p < 0.05. T-tests or One-way ANOVA with Tukey post-hoc were run to compare groups [ ].
## Results
### Lower fraction of myosin molecules in the super-relaxed state with aberrant ATP consumption in human NEB -NM
We first assessed the proportion of myosin heads in the super-relaxed state in human control and NM samples. Since this conformational state strongly correlates with the rate of ATP consumption in resting muscle fibres [ ], we used a Mant-ATP chase protocol. As the scientific literature indicates that myofibres from NM patients mainly express the cardiac/skeletal slow myosin heavy chain [ ], we restricted our analysis to this type of fibres. A total of 427 muscle fibres were tested (8–10 myofibres for each of the 12 controls and for each of the 37 patients—list of subjects in Additional file : Table S1). NEB -NM patients overall exhibited faster ATP consumption indicating significantly lower levels of myosin heads in the super-relaxed state when compared with controls (Fig. A–C). Despite this alteration, the actual ATP turnover time of super-relaxed myosin molecules was not affected (Fig. D, E). We repeated similar experiments in NM human tissue with mutations in the ACTA1 , TPM2 or TPM3 genes (associated with defects in actin or actin-binding proteins, Additional file : Table S1). Interestingly, ACTA1 -NM individuals displayed similar features as NEB -NM patients (Fig. A–E), whilst TPM2 -NM and TPM3 -NM were indistinguishable from controls (Fig. A–E). To gain insight into whether these alterations are specific to NEB (and ACTA1 ) gene mutations or related to NM-related histopathological changes, we studied myofibres from patients with an acquired form of the disease known as sporadic late-onset NM (SLONM). SLONM contrasts with typical genetic NM, since it occurs in the absence of any known mutations in NM-related genes. The onset of SLONM is also usually different as it starts in adulthood and progresses rapidly in a limb-girdle and axial pattern [ ]. Nevertheless, histopathologically, nemaline rods, sarcomeric disarray and reduced cellular force-generating capacity have been observed [ ]. SLONM patients did not exhibit any significant difference in the number/ATP turnover rate of myosin heads in the super-relaxed state when compared with controls, TPM2 -NM and TPM3 -NM patients (Fig. A–E). As the age range for the 37 patients with genetic or acquired NM varied between 1 and 70 years old, we tested whether our results are age dependent. Plotting myosin conformational state as a function of age did not reveal any long-term secondary adaptation (Fig. F).
Myosin structure and relaxed conformational states in humans. Typical Mant-ATP chase experimental data show exponential decays for muscle fibres isolated from all the different groups ( A ). The proportion of myosin molecules in the disordered-relaxed (P1, B ) and super-relaxed states (P2, C ) as well as their respective ATP turnover lifetimes (T1, D and T2, E ) are presented. F Shows the P1 data as a function of age. By staining and imaging myosin using super-resolution microscopy ( G , scale bar = 10 µm) myosin filament length is calculated ( H ). Dots are individual subject’s average data. Means and standard deviations also appear on histograms. *Denotes a difference ( p < 0.05) when compared with controls (CTL)
To further evaluate if the changes in the presence of NEB mutations are due to disarray of myosin filaments, we used super-resolution microscopy followed by DDecon analysis. Thirty-five myofibres were tested from four controls and three NEB -NM patients (5 fibres per subject). Regular striated arrays of myosin filaments were observed for both patients and controls (Fig. G). Additionally, the length of these filaments had ranges that were consistent with the inter-individual and inter-muscle heterogeneity reported previously (Fig. H) [ ].
These results indicate that, in the presence of NEB mutations, myosin heads are not properly sequestered (onto the myosin filament backbone) in resting skeletal muscle, consuming unusually large quantities of ATP. Our results also suggest that these alterations are not specific to NEB mutations but rather to myofilament-linked mechanisms that are common with ACTA1 mutations. NM-associated histopathological disruptions such as the presence of nemaline rods may not be sufficient to drive the myosin maladaptations.
#### The decreased number of super-relaxed myosin molecules may be linked to some alterations affecting myosin-binding proteins in human NEB -NM and in a nebulin conditional knockout mouse model
Myosin-binding protein C (MyBP-C) acts as a linker for myofilaments (between actin and myosin filaments); its role in stabilizing the myosin super-relaxed state has recently been highlighted by mutations in its core leading to deleterious hypertrophic cardiomyopathy [ ]. Cardiac MyBP-C has then been extensively studied, whilst skeletal MyBP-C requires further attention [ ]. As a proof-of-concept, to explore MyBP-C’s potential functional role in the increased disordered-relaxed state of myosin heads in NEB -NM, we first partially ablated the endogenous MyBP-C [ , ] by using a published protocol consisting of soaking individual muscle fibres in an extracting buffer for 1-h at room temperature [ , ]. The precise amount of MyBP-C ablated is thought to be more than 70% [ , ] but was not directly evaluated in the present study as we did not get reproducible western blot/antibody data from single muscle fibres. A total of 91 myofibres were tested from five controls and five NEB -NM patients (8–10 fibres per subject). With the Mant-ATP chase protocol, MyBP-C partial ablation significantly decreased the number of myosin heads in the super-relaxed state in controls but not in NEB -NM patients (Fig. A, B). Hence, MyBP-C partial absence may alleviate the differences between NEB -NM patients and controls. Besides MyBP-C, other myosin-binding proteins may be involved. Myosin regulatory light chains (RLC) bind to the lever arm region of myosin heads and play an important role in maintaining the integrity of the super-relaxed state [ ]. To, once again, explore whether RLC may also contribute to the changes seen in NEB -NM patients, we extracted the endogenous RLCs by incubating myofibres in a well-recognized extracting buffer for 30 min at 4 °C [ ]. As for MyBP-C, the exact level of RLC extracted is thought to be more than 90% [ ], nevertheless, we did not assess it here as we did not have reliable western blot/antibody results from individual myofibres. Fibres going through this process were then thoroughly washed with the rigor solution before running Mant-ATP chase experiments. A total of 72 myofibres were tested from five controls and five NEB -NM patients (7 to 8 fibres per subject). As for MyBP-C partial ablation, RLC partial extraction significantly lowered the proportion of myosin molecules in the super-relaxed state in controls but not in NEB -NM patients (Fig. A, B), indicating that RLC partial ablation may reduce the differences between NEB -NM patients and controls. As myofilament proteins are subject to multiple post-translational modifications impacting their function (especially MyBP-C and RLC), we explored whether modulating the phosphorylation status of the myofilament proteins would have effects on myosin head conformation in the patients. For that, we incubated myofibres in a lambda phosphatase solution for 1-h at room temperature [ ]. The extent to which the lambda phosphatase solution lowers phosphorylation in individual myosin/myosin-binding proteins remains unclear as in the present study, we did not run western blots confirming the dephosphorylation. In total, 86 muscle fibres were used from five controls and five NEB -NM patients (8–10 fibres per subject). Mant-ATP chase experiments then revealed that the phosphatase treatment increased the proportion of myosin molecules in the super-relaxed conformation in NEB -NM patients (Fig. A, B). Importantly, the lambda phosphatase treatment dampened the differences observed between NEB -NM patients and controls.
Modulation of Myosin Regulatory light chains (RLC) and Myosin-binding protein C (MyBP-C) levels and phosphorylations in humans and mice. The proportion of myosin heads in the disordered-relaxed (P1, A ) and super-relaxed states (P2, B ) are presented for humans. Dots are individual subject’s average data. C Is a typical super-resolution image (A, scale bar = 10 µm), and resultant MyBP-C filament length ( D ). Additionally, the proportion of myosin molecules in the disordered-relaxed (P1, E ) and super-relaxed states (P2, F ) for wild-type (WT) and transgenic (cNeb KO) mice are shown. Dots are individual mouse’s average data. Means and standard deviations also appear on all histograms. *Denotes a significant difference ( p < 0.05) when compared with controls/WT with similar treatment. # refers to a significant difference ( p < 0.05) when compared with before treatment for similar group (CTL/NEB-NM or WT/cNeb KO)
As MyBP-C and RLC may have some functional implications in the disrupted super-relaxed state of NEB -NM patients, we imaged MyBP-C localization/disarray by applying super-resolution microscopy and DDecon analysis. 35 myofibres were tested from four controls and three NEB -NM patients (5 fibres per individual). Similar to myosin filaments, we observed regular MyBP-C striations in both patients and controls (Fig. C). Nevertheless, strikingly, the length of each MyBP-C segment was found subtly increased in NEB -NM patients when compared with controls (Fig. D). This suggests that MyBP-C localization extends beyond the C-zone in patients. To pursue MyBP-C investigations with the leftover human tissue, we measured the global content and phosphorylation levels (S59 and T84) for slow MyBP-C using Western blotting and antibodies known to work with human muscle samples. We found tendencies towards lower total abundance (Additional file : Fig. S1A) and higher phosphorylations (Additional file : Fig. S1B-C), even though data appeared patient-specific and thus overall variable.
To validate all the above human results, we intended to assess whether similar changes are recapitulated in a relevant mouse model of NM. Whilst the conventional NEB knockout model and a model in which exon 55 of the NEB gene is deleted exist, mice die within days after birth due to complex developmental defects and abnormalities [ ]. As patients with NEB mutations often survive to adulthood with considerably milder myopathic phenotypes than the two mouse models described above, to investigate the consequences of NEB mutations/decreased myosin super-relaxed state, we took advantage of a conditional nebulin KO mouse model (cNeb KO) where muscle-specific deletions are present from birth [ ]. We used 94 muscle fibres from five cNeb KO and five control mice (8 to 10 fibres per animal). We verified the presence of a myosin super-relaxed state destabilization in cNeb KO mice using the Mant-ATP chase protocol (Fig. E, F). Additional 154 mouse myofibres (5–6 fibres per mouse) were run where MyBP-C was partially ablated or RLC extracted or phosphorylation down-regulated using the same experimental protocols as for humans. Interestingly, we observed the same significant differences as for humans strengthening our findings (Fig. E, F).
Overall, our results indicate a potential role of MyBP-C deletion and/or RLC extraction and/or dephosphorylation in disrupting the myosin super-relaxed state and related ATP consumption in resting muscle fibres from NEB -NM patients and from cNeb KO mice.
#### The lower proportion of myosin heads in the super-relaxed conformation is associated with a metabolic remodeling in nebulin conditional knockout mouse model
To gain insights into the consequences on energy metabolism/usage of our findings, we pursued additional animal model experiments. We isolated individual limb (tibialis cranialis) muscle fibres from cNeb KO and control mice and ran a proteomics analysis through quantitative LC–MS/MS tandem mass spectrometry. To this end, we utilised manually dissected single fibres to reduce the influence of proteins from other tissue and cell types and to more closely correlate our findings with the above single fibre observations. We were able to quantify 617 proteins, of which, further filtration by p-value ( p < 0.05) revealed that 250 proteins were differentially expressed, of these 111 and 139 were upregulated in the cNeb KO and control mice, respectively (Fig. A, Additional file : Table S2). We generated a volcano plot to visualize differentially upregulated proteins, annotated with the top 10 most significant proteins in each experimental group. Importantly, where a protein is denoted as more highly expressed in controls, it can also be interpreted as cNeb KO mice possessing a reduction in expression. The most significantly upregulated proteins belonged to the cNeb KO group and consisted of ZASP (Z-band Alternatively Spliced PDZ-motif) or LIM domain-binding protein 3 and alpha-actinin-2 with the latter possessing the highest log2FC (Fig. B). Moreover, we observed a significant change in myosin binding proteins H and C (Fig. B–D). Further, all proteins with a log2fc > 1.5 were visualised in the heatmap in (Fig. E), which highlighted a number of proteins involved in cytoskeletal structure but also and importantly metabolic pathways. To more accurately determine functional associations, we carried out gene ontology (GO) analysis on all proteins that passed p-value ( p < 0.05) filtration using the Metascape analysis resource (Fig. A, B, Additional file : Table S3). Biological functions associated with metabolism appear distinct between cNeb KO and control muscles. A suppression of proteins involved in glucose catabolic processes was observed in cNeb KO mice when compared with controls. This was accompanied by an increase in aerobic respiration, aerobic electron transport chain and Tricarboxylic Acid Cycle (TCA) in cNeb KO mice when compared with controls. These findings, coupled with an increase in proteins associated with the transition between fast and slow fibre type pathways, indicate an alteration in ATP production in cNeb KO muscle towards higher yielding aerobic pathways. Fibres for these proteomic studies were all derived from the tibialis cranialis skeletal muscles where the fibre type is predominantly fast twitch. However, to determine whether fibre type sampling may be the underlying cause of these proteomic differences fibre type classification using myosin quantities was performed [ ]. All samples were categorised as fast glycolytic fibres (IIx/IIb) (Additional file : Table S4). Additionally, following Pearson correlation analysis (Additional file : Fig. S2), all samples were more similar based on genomic background than fibre type.
Comparative analysis of protein changes. A Venn diagram depicting detected proteins that possessed a significantly greater expression in WT (red) and cNEB KO (blue)respectively, as well as those unchanged between experimental groups. B Volcano plot displaying Log2 fold change against Log10 p -value. Dark blue dots indicate FDR (q value) < 0.05 whilst light blue dots indicate p < 0.05 and black dots indicate p > 0.05. The top 10 most significant proteins for each experimental group have been annotated in blue (cNEB KO) or red (WT) using protein names except were due to size constraints gene names were used (MRP-L27, MRCK alpha, FOXRED2, PARP1, DLST, SR-Beta and VEGFR2 (39S ribosomal protein L27-mitochondrial, Serine/threonine-protein kinase MRCK alpha, FAD-dependent oxidoreductase domain-containing protein 2, Poly[ADP-ribose] polymerase 1,2-Oxoglutarate Dehydrogenase Complex Component E2, Signal recognition particle receptor subunit beta, Vascular endothelial growth factor receptor 2, respectively). Myosin binding protein C and H were also annotated and their abundances are highlighted in the volcano plots in C , D . E A heat map was created to illustrate the proteins with the greatest fold change, all proteins included possessed a Log2 FC > 1.5. For readability gene names rather than protein names were included in the heap map the conversion between gene name and protein names can be found in Additional file : Table S2. ***Indicates p < 0.001
Ontological analysis of the proteins differentially expressed between WT and cNEB KO and proteomic differences induced following piperine administration. A Ontological associations between 111 proteins upregulated in cNEB KO (blue) and 139 proteins upregulated in WT mice (red) were established using Metascape and visualised using Cytoscape. Grey lines indicate a direct interaction, circle size is determined by enrichment and circle colour is determined by p value. Proteins upregulated in the WT mice can also be considered as down regulated in the cNEB KO. WT and cNEB KO networks were created separately with identical enrichment and p value scaling parameters. For graphical representation both WT and cNEB KO networks were scaled to match the enrichment key. The clusters with the highest enrichment are documented in the table shown in B, with the enrichment, p value and proteins present within the cluster. All full list of all clusters and proteins present is available in Additional file : Table S2 ( B ). C Venn diagram depicting detected proteins that were significantly expressed in control (purple) and following piperine administration (green), as well as those unchanged between experimental groups. Bar graphs depicting significant differences present in the three proteins which possessed significant differences between either WT and cNEB KO or between Control and Piperine administration, the two datasets were not subjected to comparative statistics. D Pie charts represent the Uniprot binding terms associated with the significantly up regulated proteins in either control or piperine administration obtained using DAVID bioinformatics database
Next, we wanted to determine whether the changes seen above in cNeb KO mice were related to the decrease in the number of myosin heads in the super-relaxed state. To achieve this, we used mouse extensor digitorum longus control muscles (this is a fast-twitch limb skeletal muscle as tibialis cranialis) and incubated them with piperine, an alkaloid found in pepper which has previously been observed to reduce the number of myosin molecules in the super-relaxed state [ , ]. After three days of incubation in 100 µM of piperine (optimal concentration to destabilise the super-relaxed state—confirmed in Fig. E, F), we again carried out LC–MS/MS tandem mass spectrometry. A total of 260 proteins were detected following low TMT exclusion. However only 42 significant protein changes were observed following p -value ( p < 0.05) filtration. The Venn diagram revealed that the 34 proteins significantly increased expression in the control group, whist only 8 were observed to have increased significantly following piperine administration (Fig. , Additional file : Table S5). Of these significant proteins, only three were also differentially expressed in the cNeb KO mice versus control database (Myosin-4, DNA-dependent protein kinase catalytic subunit and actin, alpha skeletal muscle). Due to the low number of significant differences, performing similar GO analysis as with the previous dataset was not possible, instead we aimed to determine whether there was an upregulation of proteins associated with specific protein–ligand interactions that may be ultimately responsible for the differences associated with the previously observed, chronic decrease in the myosin super-relaxed state. Indeed, while control fibres possessed an array of different ligand binding, piperine administration predominantly upregulated proteins which possessed ATP/nucleotide binding (Fig. D, Additional file : Table S6). These proteins may therefore be involved in detecting early dysregulation of ATP utilization present following the disturbance of myosin conformational states.
## Discussion
Our study is one of the first to characterise the myosin super-relaxed state in human skeletal muscle, as most of the scientific literature thus far is on cardiac tissue. We demonstrate that isolated muscle fibres from humans diagnosed with NEB -NM have a surprising destabilization of myosin super-relaxed state and excessive energy consumption. Consistent with these observations, we indicate that such ATP overconsumption has potential consequences on the myofibre proteome of a mouse NM model.
### Myosin super-relaxed state destabilization as a pathological contributor and/or a compensatory process
The super-relaxed conformation is a highly conserved and regulated state [ ]. Its dysregulation in the context of skeletal muscle diseases is a novel finding. A reduction in the super-relaxed state is involved in the aetiology of other genetic diseases of cardiac muscle, such as hypertrophic cardiomyopathy (HCM). HCM is estimated to affect at least 1 in 500 individuals and is primarily caused by mutations in genes encoding the human β-cardiac/skeletal slow myosin heavy chain ( MYH7 ) or cardiac MyBP-C ( MYBPC3 ) [ , ]. The subtle pathogenic amino acid substitutions in the β-cardiac/skeletal slow myosin heavy chain mesa region destabilize the inter-head motif area crucial for forming and preserving the super-relaxed state [ , , ], whilst variants in MYBPC3 cause truncations and haploinsufficiency in cardiac MyBP-C, releasing its restricting power on myosin heads and lowering the number of myosin molecules in the super-relaxed conformation [ ]. All these are recognised as major components of the hyper-contractile HCM pathophysiology accounting for impaired cellular relaxation and enhanced force-generating capability [ ]. Hence, here, it is reasonable to postulate that the decreased level of myosin heads in the super-relaxed state that we observed in NEB -NM (and ACTA1 -NM) patients contribute to the aetiology of hypo-contractile NM through under-appreciated metabolic changes. More precisely, its involvement may be complex and may initially be a compensatory mechanism by which muscle fibres have more myosin heads available for actin binding to account for the depressed actin filament activation and cellular force-producing capacity [ , , , ]. Indeed, disordering of myosin heads is proposed to facilitate the interaction of myosin with actin [ ]. These interactions would be in weakly bound states that do not generate force but would contribute to stiffness [ ]. In the long-term, this contractile over-compensation may become detrimental. Myosin super-relaxed state destabilization in NEB -NM (and ACTA1 -NM) patients may have major consequences on ATP consumption and muscle metabolism, straining energy resources. This would be in line with the glycogen deposition and misshapen mitochondria observed in some patients and in most of the mouse models [ , ].
### Consequences: metabolic reprogramming when the myosin super-relaxed state is downregulated
Although more comprehensive studies are warranted, shifting myosin heads away from their super-relaxed conformation means excessive energy consumption and most likely explains the profound abnormalities in energy usage seen in NEB -NM (and ACTA1 -NM) patients’ muscle biopsy specimens [ , ]. Skeletal muscle depends on a large number of pathways to produce ATP with cellular respiration being the most efficient machinery, supplying more than 90% of the basal energy requirements [ ]. In the present study, we observed, in the presence of abnormal nebulin content, a metabolic reprogramming consisting of a shift away from glycolytic pathways to mitochondrial oxidative phosphorylation to meet the increased energy demands. This may have potential whole-body consequences. On average, adult humans utilise 8 MJ day . Most of this utilisation, known as the basal metabolic rate, is required for basic cellular functions. Even though the resting skeletal muscle metabolic rate per unit volume is low (0.5 W kg ), it accounts for approximately 25% of the obligatory whole-body thermogenesis [ ]. Here, the disordered myosin heads in patients may generate a greater overall thermogenesis [ ]. Shifting myosin heads away from their super-relaxed conformation by as little as 10% may induce an increase in thermogenesis and energy usage by 0.7 MJ day [ ]. Over a period of a year this would lead to a weight loss of 7 kg of fat [ ]. Shifting heads towards a disordered conformation by 20%, as found in the present work, would double skeletal muscle thermogenesis and would increase the whole-body basal metabolic rate by 16% [ ]. This would explain clinical findings reporting NM patients being lean or underweight.
### Causes: myosin-binding protein disruption as a potential contributor of the decreased number of super-relaxed myosin molecules
In contrast to HCM, in the present study, the downregulation of the super-relaxed conformation in NEB -NM (and ACTA1 -NM) cannot be attributed to the mutations but rather to indirect processes that could interfere with the levels of myofilament proteins. Our proteomics analysis has confirmed reductions of fast MyBP-C and fast RLC contents together with an up-regulation of myosin essential light chains (ELC) 1/3 in the presence of nebulin mutation. Thus, here, we explored the potential roles of myosin-binding proteins in NEB -NM. We observed significant functional differences when MyBP-C is partially ablated, RLC extracted or myofilament dephosphorylated. According to the literature, modulating the numbers of cardiac RLC or MyBP-C modifies the number of myosin molecules in the super-relaxed state by destabilizing the thick filaments, untethering myosin heads [ , , ]. Moreover, when comparing the phosphorylated state of cardiac MyBP-C to its dephosphorylated state, it has been shown that the phosphorylated state promotes a higher myosin order whilst the phosphomimetic state favours disordered myosin indicative of a decreased proportion of myosin heads in the super-relaxed state [ ]. Considering all these findings, it is tempting to suggest that RLC or MyBP-C are involved in the depression of the super-relaxed conformation in NEB -NM. The low number of patients tested for our MyBP-C- and RLC-related experiments as well as the absence of precise characterisations of MyBP-C and RLC deletions in our functional assays are obvious limitations here. Hence, further studies specifically focusing their attention on these aspects are required.
## Conclusion
Taken together, our data show that, in resting muscle fibres from NEB -NM patients, the myosin-stabilizing conformational state is disrupted. Our findings also suggest that the subsequent significant increase in basal ATP consumption leads to a modification of the myofibre proteome, more specifically of energy proteins. Our results then give new unexpected insights into unexplained NEB -NM pathological features, namely odd appearance of energetic proteins, and further highlight the potential benefits of drugs targeting myosin activity in NM patients.
## Supplementary Information
|
This is the first description of slowly progressive Niemann-Pick disease type C (NPC) without the typical lysosomal storage in bone marrow and viscera in two descendants of a group of 17th century French-Canadians. The index patient was a married 43-year-old woman with onset of dementia in her thirties, later followed by the development of ataxia and athetoid movements. Her autopsy disclosed frontal lobe atrophy, neurolysosomal storage with oligolamellar inclusion and tau-positive neurofibrillary tangles. Of the 119 family members screened, only a married 42-year-old sister displayed symptoms of a dementia. Both women displayed vertical supranuclear ophthalmoplegia; expressive aphasia; concrete, stimulus-bound, perseverative behavior; and impaired conceptualization and planning. Cultured fibroblasts showed decreased cholesterol esterification and positive filipin staining, but no mutation was detected in coding or promoter regions of the NPC1 gene using conformation sensitive gel electrophoresis and sequencing. Sequencing showed a homozygous gene mutation that is predicted to result in an amino acid substitution, V39M, in the cholesterol binding protein HE1 (NPC2). Adult-onset NPC2 with lysosomal storage virtually restricted to neurons represents a novel phenotypic and genotypic variant with diffuse cognitive impairment and focal frontal involvement described for the first time. |
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. Accumulating evidence has shown that 43kDa TAR-DNA-binding protein (TDP-43) is the disease protein in ALS and frontotemporal lobar degeneration. We previously reported a familial ALS with Bumina bodies and TDP-43-positive skein-like inclusions in the lower motor neurons; these findings are indistinguishable from those of sporadic ALS. In three affected individuals in two generations of one family, we found a single base-pair change from A to G at position 1028 in TDP-43, which resulted in a Gln-to-Arg substitution at position 343. Our findings provide a new insight into the molecular pathogenesis of ALS. |
The timing and yield of metabolic studies for patients with neurodevelopmental disorders is a matter of continuing debate. We determined the yield of additional or repeated metabolic studies in patients with neurodevelopmental disorders. Patients referred to a tertiary diagnostic center for patients with unexplained neurodevelopmental disorders were included. Initial metabolic studies had been performed in most patients (87%) before referral. Additional/repeated metabolic studies were individually tailored. Twelve metabolic diseases of 433 patients studied (2.8%) were diagnosed, despite normal initial metabolic studies before referral. Specific metabolic investigations lead to a greater diagnostic yield in patients with neurodevelopmental disorders. |
## Objective
We conducted a Mendelian randomization (MR) study to disentangle the comparative effects of lipids and apolipoproteins on ischemic stroke.
## Methods
Single‐nucleotide polymorphisms associated with low‐ and high‐density lipoprotein (LDL and HDL) cholesterol, triglycerides, and apolipoprotein A‐I and B (apoA‐I and apoB) at the level of genomewide significance ( p < 5 × 10 ) in the UK Biobank were used as instrumental variables. Summary‐level data for ischemic stroke and its subtypes were obtained from the MEGASTROKE consortium with 514,791 individuals (60,341 ischemic stroke cases, and 454,450 non‐cases).
## Results
Increased levels of apoB, LDL cholesterol, and triglycerides were associated with higher risk of any ischemic stroke, large artery stroke, and small vessel stroke in the main and sensitivity univariable MR analyses. In multivariable MR analysis including apoB, LDL cholesterol, and triglycerides in the same model, apoB retained a robust effect ( p < 0.05), whereas the estimate for LDL cholesterol was reversed, and that for triglycerides largely attenuated. Decreased levels of apoA‐I and HDL cholesterol were robustly associated with increased risk of any ischemic stroke, large artery stroke, and small vessel stroke in all univariable MR analyses, but the association for apoA‐I was attenuated to the null after mutual adjustment.
## Interpretation
The present MR study reveals that apoB is the predominant trait that accounts for the etiological basis of apoB, LDL cholesterol, and triglycerides in relation to ischemic stroke, in particular large artery and small vessel stroke. Whether HDL cholesterol exerts a protective effect on ischemic stroke independent of apoA‐I needs further investigation. ANN NEUROL 2020;88:1229–1236
Blood lipids are established causal factors in the development of stroke. , , It has been shown that high concentrations of low‐density lipoprotein (LDL) cholesterol increase the risk of ischemic stroke, , , whereas high concentrations of high‐density lipoprotein (HDL) cholesterol possibly decrease the risk of ischemic stroke, particularly small vessel stroke. , Furthermore, large‐scale randomized clinical trials have revealed that lowering cholesterol concentrations with statins reduces the risk of ischemic and overall stroke, , , despite an increase in hemorrhagic stroke. However, given high phenotypic and genetic correlation across different lipids and apolipoproteins, it remains unclear whether one or more lipid‐related entities account for the observed associations between lipids and stroke. Disentangling the associations of atherogenic lipoprotein lipids and risk of stroke is of great public health and clinical importance. First, a better understanding of the comparative role of lipoprotein lipids in stroke not only facilitates a clearer perception of the underlying pathophysiology of stroke, but also helps to capture the most effective biomarker and corresponding agent that lipid‐modifying therapeutics should target. Second, the findings can provide an evidence basis in guiding the prevention and treatment of stroke among more than one‐quarter of the general population who has discordant apolipoprotein B (apoB) and LDL cholesterol levels, in particular those with obesity or type 2 diabetes. , Third, such investigation will help unify the guidelines concerning regular apoB measurement supported by European Society of Cardiology/European Atherosclerosis Society, but not by the American College of Cardiology/American Heart Association.
The amount of cholesterol and triglycerides vary largely between lipoprotein particles, , , which results in an imprecise quantification of the number of atherogenic lipoproteins, although the levels of LDL cholesterol and triglycerides quantifies the levels of these lipid substances carried in circulating lipoproteins. In contrast, one apoB molecule is included in each circulating atherogenic lipoprotein particle. , Thus, the level of apoB molecules is proportional to the number of circulating atherogenic particles in the blood. Available evidence indicates causal effects of increased LDL cholesterol, triglycerides, and apoB on increasing stroke risk , and a stronger effect of apoB compared to LDL cholesterol on cardiovascular disease. , It is plausible to assume that each lipid‐related entity played an individual causal role or that one trait, such as apoB, predominated and accounted for the associations of related lipoprotein particle entities. Confined by potential methodological limitations, such as residual confounding and reverse causality, traditional observational study designs are unable to infer causality regarding the role of lipoprotein lipids in the development of stroke. Another approach is the Mendelian randomization (MR) design, which utilizes genetic variants as instrumental variables for an exposure to determine causality of an exposure‐outcome association. Given correlations across lipid‐related traits, the multivariable MR framework, as an extension to the traditional MR method, should be recommended to appraise the association of correlated multiple risk factors with the outcome of interest simultaneously. By including the genetic associations for multiple exposures in the same model, the multivariable MR can assess which traits retain causal associations with the outcome through the genetic protection against conventional biases, including unobserved confounders, reverse causality, the inherent correction for measurement error, and the avoidance of collider bias. Here, we employed the traditional MR analysis to determine the associations of individual lipid‐related traits with ischemic stroke and then multivariable to MR analysis to elucidate which of the atherogenic lipid traits accounts for the etiological basis of lipoprotein lipids in relation to stroke.
## Materials and Methods
### Study Design
An overview of the study design and used data sources are displayed in Figure and Supplementary Table . Genetic instruments for LDL and HDL cholesterol, triglycerides, and apoA‐I and apoB were selected based on the UK Biobank study. Data for the associations of the lipid‐related traits associated single‐nucleotide polymorphisms (SNPs) with ischemic stroke and subtypes were available from the MEGASTROKE consortium. The univariable MR analysis aimed to investigate the association of individual lipid‐related traits with ischemic stroke and the multivariable MR analysis aimed to compare the independent effects of correlated lipid‐related traits on ischemic stroke. We first determined which one of apoB, LDL cholesterol, and triglycerides predominantly accounted for the causal associations with ischemic stroke. We then assessed the predominant entity accounted for the inverse association of HDL‐related phenotypes with ischemic stroke (HDL cholesterol and apoA‐I). To expel the possibility of reverse causality, we performed a reverse MR analysis to examine the influence of liability to stroke on 5 lipid‐related traits. The UK Biobank study was approved by the North West Multicenter Research Ethics Committee. Original studies included in the MEGASTROKE consortium had been approved by a relevant review board. The present analyses were approved by the Swedish Ethical Review Authority.
Overview of study design. There are three key assumptions for Mendelian randomization (MR). Assumption 1: the genetic variants selected as instrumental variables should be robustly associated with the lipid‐related traits. Assumption 2: the used instrumental variables should not be associated with any potential confounders. Assumption 3: the genetic variants of an exposure should affect the risk of the outcome merely through the risk factor, not via other alternative pathways. IVW = inverse variance weighted; SNP = single‐nucleotide polymorphism.
### Genetic Instrument Selection
Genetic variants, in this case SNPs, associated with LDL and HDL cholesterol, triglycerides, and apoA‐I and apoB levels were extracted as instrumental variables for corresponding lipid‐related traits at the genomewide significance level ( p < 5 × 10 ) from the UK Biobank study including up to 343,992 individuals of European ancestry. The mean age of included participants was 56.9 years old and approximately 54% were women. The mean (standard deviation [SD]) levels were 3.57 (0.87) mmol/L for LDL cholesterol and 1.45 (0.38) mmol/L for HDL cholesterol. The median level of triglycerides was 1.50 (interquartile range = 1.11) mmol/L. The mean values for apoB and apoA‐I were 1.03 (0.24) g/L and 1.54 (0.27) g/L, respectively. Association tests were adjusted for age, sex, and a binary variable denoting the genotyping chip individuals were allocated to in UKBB. The linkage disequilibrium (LD) clumping was undertaken to select independent SNPs (r < 0.001) based on a reference panel of 503 Europeans from phase III (version 5) of the 1000 Genomes Project and the SNP with the smallest p values for the association with the trait of interest was retained in each locus. In univariable MR analysis, we used 220 SNPs as instrument variables for LDL cholesterol, 534 SNPs for HDL cholesterol, 440 SNPs for triglycerides, 440 SNPs for apoA‐I, and 255 SNPs for apoB (Supplementary Table ). By combing SNPs from related lipid‐traits and selecting independent SNPs (r < 0.01) by clump function in TwoSampleMR package, we used 548 SNPs in the multivariable MR analysis of LDL cholesterol, triglycerides and apoB, and 569 SNPs in the multivariable MR analysis of HDL cholesterol and apoA‐I. We used 32 SNPs associated with stroke at the genomewide significance level as the genetic instruments for stroke in the reverse MR analysis.
### Outcome Source
Summary‐level data for ischemic stroke and subtypes were obtained from the MEGASTROKE consortium encompassing 29 genomewide association studies (GWAS) with a final sample of 514,791 individuals (60,341 ischemic stroke cases and 454,450 non‐cases) of multi‐ancestry (European people as the majority, 86%). The stroke cases were defined as rapidly developing signs of focal (or global) disturbance of cerebral function, lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin. Any ischemic stroke was defined by all stroke cases except for intracerebral hemorrhage. Any ischemic stroke included large artery ischemic stroke (LAS; 6,688 cases), cardioembolic ischemic stroke (CES; 9,006 cases), and small vessel ischemic stroke (SVS; 11,710 cases) according to the Trial of Org 10,172 in Acute Stroke Treatment criteria and also included ischemic stroke of undefined subtype. Association test was performed under an additive genetic model with a minimum of sex and age as covariates. Summary‐level data for stroke based merely on European population were used in a sensitivity analysis. Summary‐level genetic data for lipids and apolipoprotein were obtained from Neale Laboratories ( ).
### Statistical Analysis
The inverse‐variance weighted method was used as major analysis. This method provides an estimate with the highest power and rely on the assumption that all SNPs are valid instrumental variables. The I (%) statistic was calculated to assess the heterogeneity among estimates across individual SNPs. The weighted median approach and MR‐Egger regression were used as secondary analyses to examine the robustness of the results and correct for pleiotropy. The weighted median analysis can generate consistent estimates if at least 50% of the weight in the analysis comes from valid instrumental variables. The MR‐Egger regression approach can detect and correct for directional pleiotropy albeit with compromised power. Given genetic and phenotypic correlations across lipid‐related traits (Pearson's R ranging from −0.49 to 0.96; Supplementary Table ), we further used multivariable inverse‐variance weighted method to disentangle and compare the effects of correlated lipid‐traits on ischemic stroke and subtypes. Odd ratios (ORs) and corresponding 95% confidence intervals (CIs) for outcomes were scaled to one‐SD increase in levels of lipid‐related traits. To account for multiple testing, we considered associations with p values below 0.003 (where p = 0.05/20 [5 lipid‐related traits and 4 stroke outcomes]) to represent strong evidence of causal associations, and associations with p values below 0.05 but above 0.003 as suggestive evidence of associations in the univariable MR analysis. The multiple testing was not tailed for multivariable MR analysis due to the mutual adjustment nature of multivariable MR analysis. Statistical power was estimated using a webtool and the results are shown in Supplementary Table . All analyses were performed using the mrrobust package in Stata/SE 15.0 (Stata Statistical Software: Release 15; StataCorp LLC, College Station, TX, USA) and the TwoSampleMR and Mendelian Randomization package in R Software 3.6.0 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2019; ).
### Data Availability
The datasets analyzed in this study are publicly available summary statistics. Data used can be obtained through cited papers.
## Results
### Univariable MR Analysis
Genetically predicated increased levels of apoB, LDL cholesterol, and triglycerides and decreased levels of apoA‐I and HDL cholesterol were significantly or suggestively associated with higher risk of acute ischemic stroke (AIS), LAS, and SVS, but not with CES. The effect sizes of the lipid‐related traits were similar for LAS and SVS than for AIS (Fig ). Results of sensitivity analyses are displayed in Supplementary Table . The observed associations persisted based on data from individuals of European descent (Supplementary Table 6). We did not detect any reverse associations of genetic liability to stroke with the levels of lipids and apolipoproteins (Supplementary Table ).
Associations of lipid‐related traits with stroke and subtypes in inverse‐variance weighted model. AIS = acute ischemic stroke; CES = cardioembolic stroke; CI = confidence interval; HDL = high‐density lipoprotein; LAS = large artery stroke; LDL = low‐density lipoprotein; OR = odds ratio; SNPs = single‐nucleotide polymorphisms; SVS = small vessel stroke.
### Multivariable MR Analysis
Results of multivariable MR analysis are displayed in Figure and Figure 4. In the multivariable MR analysis with mutual adjustment for apoB, LDL cholesterol, and triglycerides, apoB retained a robust causal association with AIS, LAS, and SVS, whereas the estimate for LDL cholesterol was reversed and that for triglycerides largely attenuated. The ORs of AIS, LAS, and SVS were 1.31 (95% CI = 1.01, 1.69), 1.69 (95% CI = 0.99, 2.87), and 2.18 (95% CI = 1.14, 4.18), respectively, for one‐SD increase of apoB (Fig ). The pattern for the comparative role of apoB, LDL cholesterol, and triglycerides in ischemic stroke persisted when the analysis was confined to individuals of merely European ancestry (Supplementary Table ). In the analysis of mutual adjustment for apoA‐I and HDL cholesterol, the magnitude of the associations for HDL cholesterol persisted or became stronger but became nonsignificant. Associations for apoA‐I became weaker or attenuated substantially to the null in the analyses of LAS and SVS. The results were consistent based on data derived from individuals of European descent and multi‐ancestries (Fig and Supplementary Table ). We did not include CES in Figures and because no association was detected in the univariable MR analysis of lipids and apolipoproteins with CES.
Associations of apolipoprotein B, LDL cholesterol, and triglycerides with stroke and subtypes in multivariable inverse‐variance weighted model. AIS = acute ischemic stroke; CI = confidence interval; LAS = large artery stroke; LDL = low‐density lipoprotein; OR = odds ratio; SNPs = single‐nucleotide polymorphisms; SVS = small vessel stroke.
Associations of apolipoprotein A‐I and LDL cholesterol with stroke and subtypes in multivariable inverse‐variance weighted model. AIS = acute ischemic stroke; CI = confidence interval; HDL = high‐density lipoprotein; LAS = large artery stroke; OR = odds ratio; SNPs = single‐nucleotide polymorphisms; SVS = small vessel stroke.
## Discussion
### Principal Findings
The present study confirmed the causal effects of apoB, apoA‐I, LDL and HDL cholesterol, and triglycerides on ischemic stroke. Results of multivariable MR analyses showed that the effect of apoB on ischemic stroke remained robust, whereas the associations of LDL cholesterol and triglycerides entities with ischemic stroke attenuated markedly to the null after the adjustment. This suggests that apoB is the critical entity that underlies the positive associations of lipid‐related factors and ischemic stroke, in particular, large artery and small vessel stroke. The associations for HDL cholesterol and apoA‐I became nonsignificant after adjustment; however, the magnitude of the associations for HDL cholesterol remained or became stronger. Whether HDL cholesterol had predominant effects on ischemic stroke needs more study.
Our findings of the univariable MR investigation are overall in line with previous studies on LDL and HDL cholesterol in relation to ischemic stroke. , , , , However, a previous MR study based on a smaller sample size revealed that only LDL cholesterol was statistically significantly associated with LAS and that HDL cholesterol was related to SVS. That study did not observe any associations of triglycerides with ischemic stroke and subtypes, which is not consistent with the present study and a recently published MR study. Discrepancy might be caused by inadequate power due to limited phenotypic variance explained by used genetic variants for the lipid trait and/or small sample size for stroke outcomes. Observational and genetic studies have reported an inverse association of apoA‐I and a positive association of apoB with ischemic stroke. , The present MR study confirmed those associations but extended the evidence to show that only apoB showed independent effects on stroke.
Studies on comparative effects of apoB, LDL cholesterol, and triglycerides on stroke are limited. Nevertheless, the finding of a predominant role of apoB in ischemic stroke observed in our study showed agreement with several studies on ischemic cardiovascular disease. , , In the Copenhagen City Heart Study, even though apoB was not found to predict ischemic stroke better than LDL cholesterol in a clear pattern, women with higher levels of apoB had similar risk estimates for ischemic cerebrovascular disease and ischemic stroke compared with those with lower levels. Notably, the observed dominant role of apoB does not discredit the causal roles of LDL cholesterol or triglycerides in ischemic stroke, as both LDL cholesterol and triglycerides are enveloped in atherogenic lipoproteins, each containing an apolipoprotein B molecule that cannot occur in physiological isolation. Instead, our study provides genetic evidence that apoB is the necessary element in order for bad lipoprotein lipids to exert their causal effect on ischemic stroke. In other words, changes in the amount of cholesterol and triglycerides in lipoproteins that are not accompanied by commensurate changes in number of lipoprotein particles containing apoB may not affect ischemic stroke risk. Mechanically, this finding is supported by the “response to retention” hypothesis that apoB is the necessary entity for atherosclerosis to occur. , In detail, particles containing apoB trapped in the tunica intima of the arterial wall cause atherosclerosis.
Studies on comparative effects of HDL cholesterol and apoA‐I are limited. The present study found a stronger effect of HDL cholesterol than of apoA‐I on ischemic stroke. However, due to an inadequate power embedding in multivariable MR analysis, whether HDL cholesterol plays a predominant protective role in the etiology of ischemic stroke needs more investigation. High HDL cholesterol levels prevent the oxidation of LDL cholesterol and increase the reverse transport of LDL cholesterol from peripheral tissues to the liver where degradation happens. These functions of HDL cholesterol lower the risk of atherogenesis and may explain why high HDL cholesterol levels reduce the risk of ischemic stroke.
### Public Health and Clinical Implication
Clinical trials have demonstrated that modifying LDL cholesterol and triglycerides through angiopoietin‐like proteins 4 and proprotein convertase subtilisin/kexin type 9 inhibitor might be promising approaches to lower the risk of ischemic stroke. , The present study supports these current treatments. More importantly, our findings shed new light on the focus of lipid‐modifying therapies, which should be the reduction in the number of atherogenic lipoprotein particles rather than the reduction in the amount of cholesterol or triglycerides within the particles. In addition, from the preventive perspective and especially among individuals with discordant apoB and LDL cholesterol levels, we promote apoB measurement as one of routine blood lipid examination.
### Strengths and Limitations
There are strengths of the present study. The major one was the multivariable MR method, which compared the roles of different correlated lipid‐related traits in ischemic stroke and exempted the findings from residual confounding and reverse causality. We used updated genetic instruments for lipid‐related traits, thereby ensuring an adequate power in analysis. The major limitation was that there were missing SNPs that might compromise the power and accuracy of analysis. However, the missing rates were not dissatisfying for AIS, LAS, and CES (all under 15%), except for SVS. Thus, the observed associations for SVS needs to be verified. In addition, a small proportion of stroke cases were from non‐European descents (around 14%), which might introduce population stratification bias. Nevertheless, the consistent findings based on data from individuals of only European ancestry indicated that there was a neglectable chance of population stratification bias twisting our findings. The CIs in the multivariable MR analysis were wide, which might reveal some degree of compromise of the precision of MR statistical model fitting strongly correlated exposures. Finally, the associations of SNPs with levels of lipid‐traits were derived from non‐fasting blood samples, which might cause inaccuracy in estimation. Nonetheless, the GWAS for lipid‐related traits found that adjustment for fasting time led to negligible alterations in the effect estimates.
## Conclusions
In summary, the present MR study provides evidence supporting apoB as the predominant trait that accounts for the etiological basis of apoB, HDL cholesterol, and triglycerides in relation to ischemic stroke, in particular large artery and small vessel stroke. Whether HDL cholesterol exerts protective effects on ischemic stroke independent of apoA‐I needs further investigation.
## Author Contributions
S.Y. and S.C.L. contributed to the conception and design of the study. S.Y. contributed to the acquisition and analysis of data. S.Y., B.T., J.Z., and S.C.L. contributed to drafting the text and preparing the figures.
## Potential Conflicts of Interest
The authors declared no conflicts of interest.
## Data Availability
The datasets analyzed in this study are publicly available summary statistics.
## Supporting information
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Serotonin is a neuromodulator that is extensively entangled in fundamental aspects of brain function and behavior. We present a computational view of its involvement in the control of appetitively and aversively motivated actions. We first describe a range of its effects in invertebrates, endowing specific structurally fixed networks with plasticity at multiple spatial and temporal scales. We then consider its rather widespread distribution in the mammalian brain. We argue that this is associated with a more unified representational and functional role in aversive processing that is amenable to computational analyses with the kinds of reinforcement learning techniques that have helped elucidate dopamine's role in appetitive behavior. Finally, we suggest that it is only a partial reflection of dopamine because of essential asymmetries between the natural statistics of rewards and punishments. |
Although often considered as a group, spinal motor neurons are highly diverse in terms of their morphology, connectivity, and functional properties and differ significantly in their response to disease. Recent studies of motor neuron diversity have clarified developmental mechanisms and provided novel insights into neurodegeneration in amyotrophic lateral sclerosis (ALS). Motor neurons of different classes and subtypes--fast/slow, alpha/gamma--are grouped together into motor pools, each of which innervates a single skeletal muscle. Distinct mechanisms regulate their development. For example, glial cell line-derived neurotrophic factor (GDNF) has effects that are pool-specific on motor neuron connectivity, column-specific on axonal growth, and subtype-specific on survival. In multiple degenerative contexts including ALS, spinal muscular atrophy (SMA), and aging, fast-fatigable (FF) motor units degenerate early, whereas motor neurons innervating slow muscles and those involved in eye movement and pelvic sphincter control are strikingly preserved. Extrinsic and intrinsic mechanisms that confer resistance represent promising therapeutic targets in these currently incurable diseases. |
There is a pressing need for objective, quantifiable outcome measures in intervention trials for children with autism spectrum disorder (ASD). The current study investigated the use of eye tracking as a biomarker of treatment response in the context of a pilot randomized clinical trial of treatment for young children with ASD. Participants included 28 children with ASD, aged 18-48 months, who were randomized to one of two conditions: Pivotal Response Intervention for Social Motivation (PRISM) or community treatment as usual (TAU). Eye-tracking and behavioral assessment of developmental functioning were administered at Time 1 (prior to randomization) and at Time 2 (after 6 months of intervention). Two well-established eye-tracking paradigms were used to measure social attention: social preference and face scanning. As a context for understanding relationships between social attention and developmental ability, we first examined how scanning patterns at Time 1 were associated with concurrent developmental functioning and compared to those of 23 age-matched typically developing (TD) children. Changes in scanning patterns from Time 1 to Time 2 were then compared between PRISM and TAU groups and associated with behavioral change over time. Results showed that the social preference paradigm differentiated children with ASD from TD children. In addition, attention during face scanning was associated with language and adaptive communication skills at Time 1 and change in language skills from Time 1 to Time 2. These findings highlight the importance of examining targeted biomarkers that measure unique aspects of child functioning and that are well-matched to proposed mechanisms of change. Autism Research 2019, 12: 779-793. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Biomarkers have the potential to provide important information about how and why early interventions effect positive change for young children with ASD. The current study suggests that eye-tracking measures of social attention can be used to track change in specific areas of development, such as language, and points to the need for targeted eye-tracking paradigms designed to measure specific behavioral changes. Such biomarkers could inform the development of optimal, individualized, and adaptive interventions for young children with ASD. |
Alterations in the gut microbiota may influence gastrointestinal (GI) dysbiosis frequently reported in individuals with autism spectrum disorder (ASD). In this study, we sequenced the bacterial 16S rRNA gene to evaluate changes in fecal microbiota between 48 children with ASD and 48 healthy children in China. At the phylum level, the number of Firmicutes, Proteobacteria, and Verrucomicrobia decreased in children with ASD, while the Bacteroidetes/Firmicutes was significantly higher in autistic children due to enrichment of Bacteroidetes. At the genus level, the amount of Bacteroides, Prevotella, Lachnospiracea_incertae_sedis, and Megamonas increased, while Clostridium XlVa, Eisenbergiella, Clostridium IV, Flavonifractor, Escherichia/Shigella, Haemophilus, Akkermansia, and Dialister decreased in children with ASD relative to the controls. Significant increase was observed in the number of species synthesizing branched-chain amino acids (BCAAs), like Bacteroides vulgatus and Prevotella copri, while the numbers of Bacteroides fragilis and Akkermansia muciniphila decreased in children with ASD compared to the controls. Most importantly, the highest levels of pathogenic bacteria were different for each child with ASD in this cohort. We found that only one functional module, cellular antigens, was enriched in children with ASD, and other pathways like lysine degradation and tryptophan metabolism were significantly decreased in children with ASD. These findings provide further evidence of altered gut microbiota in Chinese ASD children and may contribute to the treatment of patients with ASD. LAY SUMMARY: This study characterized the gut bacteria composition of 48 children with ASD and 48 neurotypical children in China. The metabolic disruptions caused by altered gut microbiota may contribute significantly to the neurological pathophysiology of ASD, including significant increases in the number of species synthesizing BCAAs, and decreases in the number of probiotic species. These findings suggest that a gut microbiome-associated therapeutic intervention may provide a novel strategy for treating GI symptoms frequently seen in individuals with ASD. Autism Res 2020, 13: 1614-1625. © 2020 International Society for Autism Research, Wiley Periodicals, Inc. |
Autism and specific language impairment (SLI) are developmental disorders that, although distinct by definition, have in common some features of both language and social behavior. The goal of this study was to further explore the extent to which specific clinical features of autism are seen in SLI. The children with the two disorders, matched for non-verbal IQ, were compared on the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS). In the SLI group, 41% met autism or autism spectrum cut-offs for social or communication domains either on the ADI or ADOS or both. No relationship was found between the language deficits exhibited by the children with SLI and their scores on the ADI and ADOS. These findings contribute to evidence that there is some overlap in social and communicative deficits between autism and SLI, supporting the view that autism and SLI share etiologic factors. This continuum of pathology between SLI and autism appears to range from structural language abnormalities as seen in individuals with SLI to individuals with SLI with both structural and social abnormalities to individuals with autism with pragmatic impairment and language abnormalities. |
Stress, especially chronic stress, is one of the most important factors responsible for precipitation of affective disorders in humans. The animal models commonly used in the investigation of stress effects are based mainly on powerful physical stressors. In the majority of cases, these models are not relevant to situations that human beings encounter in everyday life. In our study, an animal model for chronic social stress has been developed for rats using a resident-intruder paradigm. This paradigm is considered a model of social defeat or subordination, and therefore may mimic situations occurring in humans. Rats were subjected daily to subordination stress for a period of five weeks and, in parallel, tested with a battery of behavioural tests. Chronically stressed rats showed behavioural changes, including decreased motility and exploratory activity, increased immobility in a forced swim test, and reduced preference for sweet sucrose solution (anhedonia). Reduced locomotor and exploratory activity represents a loss of interest in new stimulating situations, implying a deficit in motivation. Increased immobility in the forced swim test indicates behavioural despair, a characteristic of depressive disorders. Decreased sucrose preference may indicate desensitisation of the brain reward mechanism. Since anhedonia is one of the core symptoms of depression in humans, our findings suggest that the rat chronic social stress model may be an appropriate model for depressive disorders. |
In human parietal cortex, the retinal location of a just seen visual stimulus is updated from one hemisphere to the other, when a horizontal eye movement brings the representation of the stimulus into the opposite visual hemifield. The present study aimed to elucidate the time course of this process. Twelve subjects performed an updating task, in which a filled circle was shown before a horizontal saccade, requiring updating of stimulus location, and a control task without visual stimulation before the saccade. Electroencephalogram (EEG) and electrooculogram (EOG) were recorded while subjects performed the tasks and LORETA source analysis was performed on event-related potential (ERP) components. ERP amplitudes were more positive in the updating condition in comparison to the control condition in two latency windows. An early positive wave starting at about 50 ms after saccade offset and originating in the posterior parietal cortex contralateral to saccade direction probably reflects the integration of saccade-related and visual information and thus the updating process. A shift of the representation of the to-be-updated stimulus to the opposite hemisphere is reflected in a later component starting approximately 400 ms after saccade offset, which is related to memory and originates in the PPC ipsilateral to saccade direction and thus contralateral to the spatial location of the updated visual stimulus. |
Chronic psychosocial stress has been suggested as "second hit" in the etiology of neuropsychiatric disease, but experimental evidence is scarce. We employed repetitive social defeat stress in juvenile mice, housed individually or in groups, and measured sensorimotor gating by pre-pulse inhibition (PPI), a marker of neuronal network function. Using the resident-intruder paradigm, 28-day old C57BL/6NCrl mice were subjected daily for 3 weeks to social defeat. PPI and basic behaviour were analyzed 10 weeks later. Whereas stress increased the level of anxiety in all animals, persistent PPI deficits were found only in individually housed mice. Thus, social support in situations of severe psychosocial stress may prevent lasting impairment in basic information processing. |
Episodic memory and episodic future thinking activate a network of overlapping brain regions, but little is known about the mechanism with which the brain separates the two processes. It was recently suggested that differential activity for memory and future thinking may be linked to differences in the phenomenal properties (e.g., richness of detail). Using functional magnetic resonance imaging in healthy subjects and a novel experimental design, we investigated the networks involved in the imagery of future and the recall of past events for the same target occasion, i.e. the Christmas and New Year's holidays, thereby keeping temporal distance and content similar across conditions. Although ratings of phenomenal characteristics were comparable for future thoughts and memories, differential activation patterns emerged. The right posterior hippocampus exhibited stronger memory-related activity during early event recall, and stronger future thought-related activity during late event imagination. Other regions, e.g., the precuneus and lateral prefrontal cortex, showed the reverse activation pattern with early future-associated and late past-associated activation. Memories compared to future thoughts were further related to stronger activation in several visual processing regions, which accords with a reactivation of the original perceptual experience. In conclusion, the results showed for the first time unique neural signatures for both memory and future thinking even in the absence of differences in phenomenal properties and suggested different time courses of brain activation for episodic memory and future thinking. |
The stress response is a multifaceted physiological reaction that engages a wide range of systems. Animal studies examining stress and the stress response employ diverse methods as stressors. While many of these stressors are capable of inducing a stress response in animals, a need exists for an ethologically relevant stressor for female rats. The purpose of the current study was to use an ethologically relevant social stressor to induce behavioral alterations in adult female rats. Adult (postnatal day 90) female Wistar rats were repeatedly exposed to lactating Long Evans female rats to simulate chronic stress. After six days of sessions, intruder females exposed to defeat were tested in the sucrose consumption test, the forced swim test, acoustic startle test, elevated plus maze, and open field test. At the conclusion of behavioral testing, animals were restrained for 30 min and trunk blood was collected for assessment of serum hormones. Female rats exposed to maternal aggression exhibited decreased sucrose consumption, and impaired coping behavior in the forced swim test. Additionally, female rats exposed to repeated maternal aggression exhibited an increased acoustic startle response. No changes were observed in female rats in the elevated plus maze or open field test. Serum hormones were unaltered due to repeated exposure to maternal aggression. These data indicate the importance of the social experience in the development of stress-related behaviors: an acerbic social experience in female rats precipitates the manifestation of depressive-like behaviors and an enhanced startle response. |
Several studies have examined impulsive choice behavior in spontaneously hypertensive rats (SHRs) as a possible pre-clinical model for Attention-Deficit/Hyperactivity Disorder (ADHD). However, this strain was not specifically selected for the traits of ADHD and as a result their appropriateness as a model has been questioned. The present study investigated whether SHRs would exhibit impulsive behavior in comparison to their control strain, Wistar Kyoto (WKY) rats. In addition, we evaluated a strain that has previously shown high levels of impulsive choice, the Lewis (LEW) rats and compared them with their source strain, Wistar (WIS) rats. In the first phase, rats could choose between a smaller-sooner (SS) reward of 1 pellet after 10 s and a larger-later (LL) reward of 2 pellets after 30 s. Subsequently, the rats were exposed to increases in LL reward magnitude and SS delay. These manipulations were designed to assess sensitivity to magnitude and delay within the choice task to parse out possible differences in using the strains as models of specific deficits associated with ADHD. The SHR and WKY strains did not differ in their choice behavior under either delay or magnitude manipulations. In comparison to WIS, LEW showed deficits in choice behavior in the delay manipulation, and to a lesser extent in the magnitude manipulation. An examination of individual differences indicated that the SHR strain may not be sufficiently homogeneous in their impulsive choice behavior to be considered as a viable model for impulse control disorders such as ADHD. The LEW strain may be worthy of further consideration for their suitability as an animal model. |
Postnatal maternal separation (PMS) has been shown to be associated with an increased vulnerability to psychiatric illnesses in adulthood. However, the underlying neurological mechanisms are not well understood. Here we evaluated its effects on neurogenesis and tonic GABA currents of cortical layer 5 (L5) pyramidal neurons. PMS not only increased cell proliferation in the subventricular zone, cortical layer 1 and hippocampal dentate gyrus in the adult brain, but also promoted the newly generated cells to differentiate into GABAergic neurons, and PMS adult brain maintained higher ratios of GABAergic neurons in the survival of newly generated cells within 5 days immediately post PMS. Additionally, PMS increased the tonic currents at P7-10 and P30-35 in cortical L5 pyramidal cells. Our results suggest that the newly generated GABAergic neurons and the low GABA concentration-activated tonic currents may be involved in the development of psychiatric disorders after PMS. |
Postnatal overfeeding is a well-known model of early-life induced obesity and glucose intolerance in rats. However, little is known about its impact on insulin signaling in specific brain regions such as the mesocorticolimbic system, and its putative effects on dopamine-related hedonic food intake in adulthood. For this study, rat litters were standardized to 4 (small litter - SL) or 8 pups (control - NL) at postnatal day 1. Weaning was at day 21, and all tests were conducted after day 60 of life in male rats. In Experiment 1, we demonstrated that the SL animals were heavier than the NL at all time points and had decreased AKT/pAKT ratio in the Ventral Tegmental Area (VTA), without differences in the skeletal muscle insulin signaling in response to insulin injection. In Experiment 2, the standard rat chow intake was addressed using an automated system (BioDAQ, Research Diets(®)), and showed no differences between the groups. On the other hand, the SL animals ingested more sweet food in response to the 1 min tail-pinch challenge and did not develop conditioned place preference to sweet food. In Experiment 3 we showed that the SL rats had increased VTA TH content but had no difference in this protein in response to a sweet food challenge, as the NL had. The SL rats also showed decreased levels of dopamine D2 receptors in the nucleus accumbens. Here we showed that early postnatal overfeeding was linked to an altered functioning of the mesolimbic dopamine pathway, which was associated with altered insulin signaling in the VTA, suggesting increased sensitivity, and expression of important proteins of the dopaminergic system. |
The current study evaluated age differences in conditioned pain modulation using a test stimulus that provided the opportunity to evaluate changes in heat pain sensitivity, sensitization, and desensitization within the same paradigm. During this psychophysical test, pain intensity clamping uses REsponse Dependent STIMulation (REDSTIM) methodology to automatically adjust stimulus intensity to maintain a desired pain rating set-point. Specifically, stimulus intensity increases until a pre-defined pain rating (the setpoint) is exceeded, and then decreases until pain ratings fall below the setpoint, with continued increases and decreases dictated by ratings. The subjects are blinded in terms of the setpoint and stimulus intensities. Younger and older subjects completed two test sessions of two REDSTIM trials, with presentation of conditioning cold stimulation between the trials of one session but not the other. The results indicated that conditioning cold stimulation similarly decreased the overall sensitivity of younger and older subjects, as measured by the average temperature that maintained a setpoint rating of 20 (on a scale of 0-100). The conditioning stimulus also significantly enhanced sensitization following ascending stimulus progressions and desensitization following descending stimulus progressions in older subjects relative to younger subjects. Thus, older subjects experienced greater swings in sensitivity in response to varying levels of painful stimulation. These results are discussed in terms of control over pain intensity by descending central modulatory systems. These findings potentially shed new light on the central control over descending inhibition and facilitation of pain. |
Brain edema is a major contributor to poor outcome and reduced quality of life after surgical brain injury (SBI). Although SBI pathophysiology is well-known, the correlation between cerebral edema and neurological deficits has not been thoroughly examined in the rat model of SBI. Thus, the purpose of this study was to determine the correlation between brain edema and deficits in standard sensorimotor neurobehavior tests for rats subjected to SBI. Sixty male Sprague-Dawley rats were subjected to either sham surgery or surgical brain injury via partial frontal lobectomy. All animals were tested for neurological deficits 24 post-SBI and fourteen were also tested 72 h after surgery using seven common behavior tests: modified Garcia neuroscore (Neuroscore), beam walking, corner turn test, forelimb placement test, adhesive removal test, beam balance test, and foot fault test. After assessing the functional outcome, animals were euthanized for brain water content measurement. Surgical brain injury resulted in significantly elevated frontal lobe brain water content 24 and 72 h after surgery compared to that of sham animals. In all behavior tests, significance was observed between sham and SBI animals. However, a correlation between brain water content and functional outcome was observed for all tests except Neuroscore. The selection of behavior tests is critical to determine the effectiveness of therapeutics. Based on this study's results, we recommend using beam walking, the corner turn test, the beam balance test, and the foot fault test since correlations with brain water content were observed at both 24 and 72 h post-SBI. |
Development of disease modifying therapeutics for Parkinson's disease (PD), the second most common neurodegenerative disorder, relies on availability of animal models which recapitulate the disease hallmarks. Only few transgenic mouse models, which mimic overexpression of alpha-synuclein, show dopamine loss, behavioral impairments and protein aggregation. Mice overexpressing human wildtype alpha-synuclein under the Thy-1 promotor (Thy1-aSyn) replicate these features. However, female mice do not exhibit a phenotype. This was attributed to a potentially lower transgene expression located on the X chromosome. Here we support that female mice overexpress human wildtype alpha-synuclein only about 1.5 fold in the substantia nigra, compared to about 3 fold in male mice. Since female Thy1-aSyn mice were shown previously to exhibit differences in corticostriatal communication and synaptic plasticity similar to their male counterparts we hypothesized that female mice use compensatory mechanisms and strategies to not show overt motor deficits despite an underlying endophenotype. In order to unmask these deficits we translated recent findings in PD patients that sensory abnormalities can enhance motor dysfunction into a novel behavioral test, the adaptive rotating beam test. We found that under changing sensory input female Thy1-aSyn mice showed an overt phenotype. Our data supports that the integration of sensorimotor information is likely a major contributor to symptoms of movement disorders and that even low levels of overexpression of human wildtype alpha-synuclein has the potential to disrupt processing of these information. The here described adaptive rotating beam test represents a sensitive behavioral test to detect moderate sensorimotor alterations in mouse models. |
Functional interaction between cannabinoid and serotonin neuronal systems have been reported in different tasks related to memory assessment. The present study investigated the effect of serotonin 5-HT4 agents into the dorsal hippocampus (the CA1 region) on spatial and object novelty detection deficits induced by activation of cannabinoid CB1 receptors (CB1Rs) using arachidonylcyclopropylamide (ACPA) in a non-associative behavioral task designed to forecast the ability of rodents to encode spatial and non-spatial relationships between distinct stimuli. Post-training, intra-CA1 microinjection of 5-HT4 receptor agonist RS67333 or 5-HT4 receptor antagonist RS23597 both at the dose of 0.016μg/mouse impaired spatial memory, while cannabinoid CB1R antagonist AM251 (0.1μg/mouse) facilitated object novelty memory. Also, post-training, intraperitoneal administration of CB1R agonist ACPA (0.005-0.05mg/kg) impaired both memories. However, a subthreshold dose of RS67333 restored ACPA response on both memories. Moreover, a subthreshold dose of RS23597 potentiated ACPA (0.01mg/kg) and reversed ACPA (0.05mg/kg) responses on spatial memory, while it potentiated ACPA response at the dose of 0.005 or 0.05mg/kg on object novelty memory. Furthermore, effective dose of AM251 restored ACPA response at the higher dose. AM251 blocked response induced by combination of RS67333 or RS23597 and the higher dose of ACPA on both memories. Our results highlight that hippocampal 5-HT4 receptors differently affect cannabinoid signaling in spatial and object novelty memories. The inactivation of CB1 receptors blocks the effect of 5-HT4 agents into the CA1 region on memory deficits induced by activation of CB1Rs via ACPA. |
Ghrelin is a peptide of 28 amino acids with a homology between species, which acts on the central nervous system to regulate different actions, including the control of growth hormone secretion and metabolic regulation. It has been suggested that central ghrelin is a mediator of behavior linked to stress responses and induces anxiety in rodents and birds. Previously, we observed that the anxiogenic-like behavior induced by ghrelin injected into the intermediate medial mesopallium (IMM) of the forebrain was blocked by bicuculline (a GABA<sub>A</sub> receptor competitive antagonist) but not by diazepam (a GABA<sub>A</sub> receptor allosteric agonist) in neonatal meat-type chicks (Cobb). Numerous studies have indicated that hypothalamic-pituitary-adrenal (HPA) axis activation mediates the response to stress in mammals and birds. However, it is still unclear whether this effect of ghrelin is associated with HPA activation. Therefore, we investigated whether anxiety behavior induced by intra-IMM ghrelin and mediated through GABA<sub>A</sub> receptors could be associated with HPA axis activation in the neonatal chick. In the present study, in an Open Field test, intraperitoneal bicuculline methiodide blocked anxiogenic-like behavior as well as the increase in plasma ACTH and corticosterone levels induced by ghrelin (30pmol) in neonatal chicks. Moreover, we showed for the first time that a competitive antagonist of GABA<sub>A</sub> receptor suppressed the HPA axis activation induced by an anxiogenic dose of ghrelin. These results show that the anxiogenic ghrelin action involves the activation of the HPA axis, with a complex functional interaction with the GABA<sub>A</sub> receptor. |
Studies using silver catfish (Rhamdia quelen) as experimental models are often applied to screen essential oils (EO) with GABAergic-mediated effects. However, the expression of GABAa receptors in the silver catfish brain remains unknown. Thus, we assessed whether silver catfish express GABAa receptor subunits associated with sedation/anesthetic process and/or neurological diseases. Additionally, we evaluated the brain expression of GABAa receptor subunits in fish sedated with Nectandra grandiflora EO and its isolated compounds, the fish anesthetic (+)-dehydrofukinone (DHF), and dehydrofukinone epoxide (DFX), eremophil-11-en-10-ol (ERM) and selin-11-en-4-α-ol (SEL), which have GABAa-mediated anxiolytic-like effects in mice. The expression of the subunits gabra1, gabra2, gabra3, gabrb1, gabrd and gabrg2 in the silver catfish brain were assessed after a 24h-sedation bath by real time PCR. Since qPCR data rarely describes mechanisms of action, which are usually found through interactions with receptors, we also performed an antagonist-driven experiment using flumazenil (FMZ). Real-time PCR detected the mRNA expression of all targeted genes in R. quelen brain. The expression of gabra1 was decreased in fish sedated with ERM; EO increased gabra2, gabra3, gabrb1 and gabrg2 expression; SEL increased gabrb1, gabrd and gabrg2 expression. EO and compounds DFX, SEL and ERM induced sustained sedation in fish and FMZ-bath prompted the recovery from ERM- and DFX-induced sedation. Our results suggest that the EO, SEL, ERM and DFX sedative effects involve interaction with the GABAergic system. Our findings support the use of the silver catfish as robust and reliable experimental model to evaluate the efficacy of drugs with putative GABAergic-mediated effects. |
This paper offers a formal account of emotional inference and stress-related behaviour, using the notion of active inference. We formulate responses to stressful scenarios in terms of Bayesian belief-updating and subsequent policy selection; namely, planning as (active) inference. Using a minimal model of how creatures or subjects account for their sensations (and subsequent action), we deconstruct the sequences of belief updating and behaviour that underwrite stress-related responses - and simulate the aberrant responses of the sort seen in post-traumatic stress disorder (PTSD). Crucially, the model used for belief-updating generates predictions in multiple (exteroceptive, proprioceptive and interoceptive) modalities, to provide an integrated account of evidence accumulation and multimodal integration that has consequences for both motor and autonomic responses. The ensuing phenomenology speaks to many constructs in the ecological and clinical literature on stress, which we unpack with reference to simulated inference processes and accompanying neuronal responses. A key insight afforded by this formal approach rests on the trade-off between the epistemic affordance of certain cues (that resolve uncertainty about states of affairs in the environment) and the consequences of epistemic foraging (that may be in conflict with the instrumental or pragmatic value of 'fleeing' or 'freezing'). Starting from first principles, we show how this trade-off is nuanced by prior (subpersonal) beliefs about the outcomes of behaviour - beliefs that, when held with unduly high precision, can lead to (Bayes optimal) responses that closely resemble PTSD. |
Diabetes mellitus induces neuropsychiatric comorbidities at an early stage, which can be ameliorated by exercise. However, the neurobiological mechanisms underlying this ameliorative effect remain unclear. The present study was conducted in Otsuka Long-Evans Tokushima fatty (OLETF) rats, which develop diabetes with age, and aimed to investigate whether social and anxiety-like behaviors and neurobiological changes associated with these behavioral phenotypes were reversed by voluntary exercise and whether those were maintained in the later stage. We investigated the effects of exercise at different diabetic stages in OLETF rats by comparing with control rats. Three groups of OLETF rats were used: sedentary rats, rats exercising on a wheel for two weeks at 4-5 weeks of age (early voluntary exercise), and those exercising at 10-11 weeks of age (late voluntary exercise). In the elevated plus-maze test, both early and late voluntary exercises did not affect anxiety-like behavior. In the social interaction tests, both early and late voluntary exercises ameliorated impaired sociability, novel exploration deficits, and hypoactivity in OLETF rats. Both early and late voluntary exercises reversed the increases in cholecystokinin-positive neuron densities in the infralimbic cortex and hippocampal cornu ammonis area 3 in the OLETF rats, although they did not affect the area-reduction in the medial prefrontal cortex and the increase in cholecystokinin-positive neuron densities in the basolateral amygdala. These suggest that voluntary exercise has therapeutic effects on impaired sociability and novel exploration deficits associated with cholecystokinin-positive neurons in specific corticolimbic regions in OLETF rats, and those are maintained after early exercise. |
Strong evidence has implicated ubiquitin signaling in the process of fear memory formation. While less abundant than ubiquitination, evidence suggests that protein SUMOylation may also be involved in fear memory formation in neurons. However, the importance of amygdala protein SUMOylation in fear memory formation has never been directly examined. Furthermore, while recent evidence indicates that males and females differ significantly in the requirement for ubiquitin signaling during fear memory formation, whether sex differences also exist in the importance of protein SUMOylation to this process remains unknown. Here we found that males and females differ in the requirement for protein SUMOylation in the amygdala during fear memory formation. Western blot analysis revealed that while females had higher resting levels of SUMOylation, both sexes showed global increases following fear conditioning. However, SUMOylation-specific proteomic analysis revealed that only females have increased targeting of individual proteins by SUMOylation following fear conditioning, some of which were heat shock proteins. This suggests that protein SUMOylation is more robustly engaged in the amygdala of females following fear conditioning. In vivo siRNA mediated knockdown of Ube2i, the coding gene for the essential E2 ligase for SUMOylation conjugation, in the amygdala impaired fear memory in males without any effect in females. Importantly, higher siRNA concentrations than what was needed to impair memory in males reduced Ube2i levels in the amygdala of females but resulted in an increase in SUMOylation levels, suggesting a compensatory effect in females that was not observed in males. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation. |
Previous studies have reported that peer groups are one of the most important predictors of adolescent and young adult marijuana use, and yet the neural correlates of social processing in marijuana users have not yet been studied. In the current study, marijuana-using young adults (n = 20) and non-using controls (n = 22) participated in a neuroimaging social exclusion task called Cyberball, a computerized ball-tossing game in which the participant is excluded from the game after a pre-determined number of ball tosses. Controls, but not marijuana users, demonstrated significant activation in the insula, a region associated with negative emotion, when being excluded from the game. Both groups demonstrated activation of the ventral anterior cingulate cortex (vACC), a region associated with affective monitoring, during peer exclusion. Only the marijuana group showed a correlation between vACC activation and scores on a self-report measure of peer conformity. This study indicates that marijuana users show atypical neural processing of social exclusion, which may be either caused by, or the result of, regular marijuana use. |
Neurodegenerative disorders of aging represent a growing public health concern. In the United States alone, there are now >5 million patients with Alzheimer's disease (AD), the most common form of dementia. No therapeutic approaches are available that alter the relentless course of AD or other dementias of aging. A major hurdle to the development of effective therapeutics has been the lack of predictive model systems in which to develop and validate candidate therapies. Animal model studies based on the analysis of transgenic mice that overexpress rare familial AD-associated mutant genes have been informative about mechanisms of familial disease, but they have not proven predictive for drug development. New approaches to disease modeling are of particular interest. Methods such as epigenetic reprogramming of patient skin fibroblasts to human induced pluripotent stem cells, which can be differentiated into patient-derived neuron subtypes, have generated significant excitement because of their potential to model more accurately aspects of human neurodegeneration. Studies focused on the generation of human neuron models of AD and frontotemporal dementia have pointed to pathologic pathways and potential therapeutic venues. This article discusses the promise and potential pitfalls of modeling of dementia disorders based on somatic cell reprogramming. |
Postural instability occurs in HIV infection, but quantitative balance tests in conjunction with neuroimaging are lacking. We examined whether infratentorial brain tissue volume would be deficient in nondemented HIV-infected individuals and whether selective tissue deficits would be related to postural stability and psychomotor speed performance. The 123 participants included 28 men and 12 women with HIV infection without dementia or alcohol use disorders, and 40 men and 43 women without medical or psychiatric conditions. Participants completed quantitative balance testing, Digit Symbol test, and a test of finger movement speed and dexterity. An infratentorial brain region, supratentorial ventricular system, and corpus callosum were quantified with MRI-derived atlas-based parcellation, and together with archival DTI-derived fiber tracking of pontocerebellar and internal and external capsule fiber systems, brain measures were correlated with test performance. The tissue ratio of the infratentorium was ~3% smaller in the HIV than control group. The HIV group exhibited performance deficits in balancing on one foot, walking toe-to-heel, Digit Symbol substitution task, and time to complete all Digit Symbol grid boxes. Total infratentorial tissue ratio was a significant predictor of balance and Digit Symbol scores. Balance scores did not correlate significantly with ventricular volumes, callosal size, or internal or external capsule fiber integrity but did so with indices of pontocerebellar tract integrity. HIV-infected individuals specifically recruited to be without complications from alcohol use disorders had pontocerebellar tissue volume deficits with functional ramifications. Postural stability and psychomotor speed were impaired and attributable, at least in part, to compromised infratentorial brain systems. |
Stress is a risk factor for the onset of mental disorders. Although stress response varies across individuals, the mechanism of individual differences remains unclear. Here, we investigated the neural basis of individual differences in response to mental stress using magnetoencephalography (MEG). Twenty healthy male volunteers completed the Temperament and Character Inventory (TCI). The experiment included two types of tasks: a non-stress-inducing task and a stress-inducing task. During these tasks, participants passively viewed non-stress-inducing images and stress-inducing images, respectively, and MEG was recorded. Before and after each task, MEG and electrocardiography were recorded and subjective ratings were obtained. We grouped participants according to Novelty seeking (NS) - tendency to be exploratory, and Harm avoidance (HA) - tendency to be cautious. Participants with high NS and low HA (n = 10) assessed by TCI had a different neural response to stress than those with low NS and high HA (n = 10). Event-related desynchronization (ERD) in the beta frequency band was observed only in participants with high NS and low HA in the brain region extending from Brodmann's area 31 (including the posterior cingulate cortex and precuneus) from 200 to 350 ms after the onset of picture presentation in the stress-inducing task. Individual variation in personality traits (NS and HA) was associated with the neural response to mental stress. These findings increase our understanding of the psychological and neural basis of individual differences in the stress response, and will contribute to development of the psychotherapeutic approaches to stress-related disorders. |
Gray matter (GM) lobar atrophy and glucose hypometabolism are well-described hallmarks of frontotemporal lobar degeneration (FTLD), but the relationships between them are still poorly understood. In this study, we aimed to show the patterns of GM atrophy and hypometabolism in a sample of 15 patients with the behavioral variant of FTLD (bv-FTD), compared to 15 healthy controls, then to provide a direct comparison between GM atrophy and hypometabolism, using a voxel-based method specially designed to statistically compare the two imaging modalities. The participants underwent structural magnetic resonance imaging and <sup>18</sup>F-fluorodeoxyglucose (FDG) positron emission tomography examinations. First, between-group comparisons of GM volume and metabolism were performed. Then, in the patient group, correlations between regional alterations and direct between-modality voxelwise comparison were performed. Finally, we examined individual patterns of brain abnormalities for each imaging modality and each patient. The observed patterns of GM atrophy and hypometabolism were consistent with previous studies. We found significant voxelwise correlations between changes in GM and FDG uptake, mainly in the frontal cortex, corresponding to the typical profile of alterations in bv-FTD. The direct comparison revealed regional variability in the relationship between hypometabolism and atrophy. This analysis revealed greater atrophy than hypometabolism in the right putamen and amygdala, and left insula and superior temporal gyrus, whereas hypometabolism was more severe than GM atrophy in the left caudate nucleus and anterior cingulate cortex. Finally, GM atrophy affected the right amygdala/hippocampus and left insula in 95 % of the patients. These findings provide evidence for regional variations in the hierarchy of hypometabolism and GM atrophy and the relationships between them, and enhance our understanding of the pathophysiology of bv-FTD. |
Dopaminergic dysfunction and changes in white matter integrity are among the most replicated findings in schizophrenia. A modulating role of dopamine in myelin formation has been proposed in animal models and healthy human brain, but has not yet been systematically explored in schizophrenia. We used diffusion tensor imaging and <sup>18</sup>F-fallypride positron emission tomography in 19 healthy and 25 schizophrenia subjects to assess the relationship between gray matter dopamine D<sub>2</sub>/D<sub>3</sub> receptor density and white matter fractional anisotropy in each diagnostic group. AFNI regions of interest were acquired for 42 cortical Brodmann areas and subcortical gray matter structures as well as stereotaxically placed in representative white matter areas implicated in schizophrenia neuroimaging literature. Welch's t-test with permutation-based p value adjustment was used to compare means of z-transformed correlations between fractional anisotropy and <sup>18</sup>F-fallypride binding potentials in hypothesis-driven regions of interest in the diagnostic groups. Healthy subjects displayed an extensive pattern of predominantly negative correlations between <sup>18</sup>F-fallypride binding across a range of cortical and subcortical gray matter regions and fractional anisotropy in rostral white matter regions (internal capsule, frontal lobe, anterior corpus callosum). These patterns were disrupted in subjects with schizophrenia, who displayed significantly weaker overall correlations as well as comparatively scant numbers of significant correlations with the internal capsule and frontal (but not temporal) white matter, especially for dopamine receptor density in thalamic nuclei. Dopamine D<sub>2</sub>/D<sub>3</sub> receptor density and white matter integrity appear to be interrelated, and their decreases in schizophrenia may stem from hyperdopaminergia with dysregulation of dopaminergic impact on axonal myelination. |
Dislipidemia is a risk factor for cognitive impairment. We studied the association between interindividual variability of plasma lipids and white matter (WM) microstructure, using diffusion tensor imaging (DTI) in 273 healthy adults. Special focus was placed on 7 regions of interest (ROI) which are structural components of cognitive neurocircuitry. We also investigated the effect of plasma lipids on cerebrospinal fluid (CSF) neurofilament light chain (NfL), an axonal degeneration marker. Low density lipoprotein (LDL) and triglyceride (TG) levels showed a negative association with axial diffusivity (AxD) in multiple regions. High density lipoproteins (HDL) showed a positive correlation. The association was independent of Apolipoprotein E (APOE) genotype, blood pressure or use of statins. LDL moderated the relation between NfL and AxD in the body of the corpus callosum (p = 0.041), right cingulum gyrus (p = 0.041), right fornix/stria terminalis (p = 0.025) and right superior longitudinal fasciculus (p = 0.020) and TG in the right inferior longitudinal fasciculus (p = 0.004) and left fornix/stria terminalis (p = 0.001). We conclude that plasma lipids are associated to WM microstructural changes and axonal degeneration and might represent a risk factor in the transition from healthy aging to disease. |
Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by absence of dystrophin protein. Dystrophin is expressed in muscle, but also in the brain. Difficulties with attention/inhibition, working memory and information processing are well described in DMD patients but their origin is poorly understood. The default mode network (DMN) is one of the networks involved in these processes. Therefore we aimed to assess DMN connectivity in DMD patients compared to matched controls, to better understand the cognitive profile in DMD. T1-weighted and resting state functional MRI scans were acquired from 33 DMD and 24 male age-matched controls at two clinical sites. Scans were analysed using FMRIB Software Library (FSL). Differences in the DMN were assessed using FSL RANDOMISE, with age as covariate and threshold-free cluster enhancement including multiple comparison correction. Post-hoc analyses were performed on the visual network, executive control network and fronto-parietal network with the same methods. In DMD patients, the level of connectivity was higher in areas within the control DMN (hyperconnectivity) and significant connectivity was found in areas outside the control DMN. No hypoconnectivity was found and no differences in the visual network, executive control network and fronto-parietal network. We showed differences both within and in areas outside the DMN in DMD. The specificity of our findings to the DMN can help provide a better understanding of the attention/inhibition, working memory and information processing difficulties in DMD. |
Fluorescent protein technology has evolved to include genetically encoded biosensors that can monitor levels of ions, metabolites, and enzyme activities as well as protein conformation and even membrane voltage. They are well suited to live-cell microscopy and quantitative analysis, and they can be used in multiple imaging modes, including one- or two-photon fluorescence intensity or lifetime microscopy. Although not nearly complete, there now exists a substantial set of genetically encoded reporters that can be used to monitor many aspects of neuronal and glial biology, and these biosensors can be used to visualize synaptic transmission and activity-dependent signaling in vitro and in vivo. In this review, we present an overview of design strategies for engineering biosensors, including sensor designs using circularly permuted fluorescent proteins and using fluorescence resonance energy transfer between fluorescent proteins. We also provide examples of indicators that sense small ions (e.g., pH, chloride, zinc), metabolites (e.g., glutamate, glucose, ATP, cAMP, lipid metabolites), signaling pathways (e.g., G protein-coupled receptors, Rho GTPases), enzyme activities (e.g., protein kinase A, caspases), and reactive species. We focus on examples where these genetically encoded indicators have been applied to brain-related studies and used with live-cell fluorescence microscopy. |
In the last decade, drastic changes in the understanding of the role of the olfactory bulb and piriform cortex in odor detection have taken place through awake behaving recording in rodents. It is clear that odor responses in mitral and granule cells are strikingly different in the olfactory bulb of anesthetized versus awake animals. In addition, sniff recording has evidenced that mitral cell responses to odors during the sniff can convey information on the odor identity and sniff phase. Moreover, we review studies that show that the mitral cell conveys information on not only odor identity but also whether the odor is rewarded or not (odor value). Finally, we discuss how the substantial increase in awake behaving recording raises questions for future studies. |
Human research with psychedelics is making groundbreaking discoveries. Psychedelics modify enduring elements of personality and seemingly reduce anxiety, depression, and substance dependence in small but well-designed clinical studies. Psychedelics are advancing through pharmaceutical regulatory systems, and neuroimaging studies have related their extraordinary effects to select brain networks. This field is making significant basic science and translational discoveries, yet preclinical studies have lagged this renaissance in human psychedelic research. Preclinical studies have a lot to offer psychedelic research as they afford tight control of experimental parameters, subjects with documented drug histories, and the capacity to elucidate relevant signaling cascades as well as conduct invasive mechanistic studies of neurochemistry and neural circuits. Safety pharmacology, novel biomarkers, and pharmacokinetics can be assessed in disease state models to advance psychedelics toward clinical practice. This chapter documents the current status of psychedelic research, with the thematic argument that new preclinical studies would benefit this field. |
This chapter discusses the premotor neural mechanisms that control horizontal saccadic eye movements. Oculomotoneurons carry a pulse-step signal that underlies the pulse-step force driving the overdamped plant. The pulse and step are both generated by a common signal, arising from medium-lead burst neurons in the pons. Their burst signal encodes saccadic eye velocity, while the number of spikes in the burst relates to the saccade amplitude. The step component, which encodes the eye position, is obtained by neural integration of the burst. Several oculomotor neural disorders can be explained by impairments in the binocular push-pull organization of this pulse-step mechanism. Plasticity of the pulse-step control, e.g., in response to muscle weakening, is mediated by cerebellar vermis and flocculus. Saccadic offset may be controlled, either by active braking, or by an exponential slide signal. The neurophysiology is summarized by a quantitative model, in which the firing rate of burst neurons is controlled by a dynamic negative feedback loop that carries the instantaneous eye position signal from the neural integrator. This signal is compared with a desired eye-position command in the head from higher centers, and the resulting dynamic motor error drives the high-gain burst cells. Instability of the system is prevented by the mutual inhibitory interaction between burst cells and omnipause neurons. The model explains many features of normal saccades, but also accounts for pathologies and abnormalities like dynamic overshoots and saccade oscillations. |
In the present study, we used a modification of the rabbit small clot embolic stroke model (RSCEM), a multiple infarct ischemia model to achieve reperfusion (REP) through the internal carotid artery (ICA) following small clot embolization. We determined if increasing regional cortical blood flow (RCBF) following an embolic stroke is beneficial to neurological outcome. We compared this to cerebral reperfusion induced by the administration of the thrombolytic Tenecteplase (TNK, 1.5 mg/kg, IV bolus) in the presence or absence of REP. In this study, we also measured the incidence of ICH following REP and thrombolytic treatment. Following embolization, RCBF was reduced to 48-55% of baseline. When REP was induced by removal of a CCA ligature, RCBF initially increased to 185% of baseline. REP (P(50)=1.18+/-0.43 mg) had no effect on embolization-induced behavior measured 24 h following embolization compared to control (P(50)=1.01+/-0.48 mg). However, TNK treatment (2-hours post-embolization) in the absence or presence of REP (initiated 2 h following embolization) significantly (p<0.05) increased the group P(50) to 2.92+/-0.55 mg and 2.42+/-0.40 mg, respectively. In addition, ICH was increased in the REP (42%, p<0.05) and REP-TNK (35%, p>0.05) group compared to either the control group (5.5%) or TNK group (10%). This study show that reperfusion of ICA can increase RCBF following embolization, but this is not associated with improved neurological outcome measured using quantal analysis. However, TNK administration significantly increased behavioral outcome when given 2 h following embolization; an increase that is not affected by combining TNK with REP. |
A significant number of studies that evaluated tactile-pain interactions employed heat to evoke nociceptive responses. However, relatively few studies have examined the effects of non-noxious thermal stimulation on tactile discriminative capacity. In this study, the impact that non-noxious heat had on three features of tactile information processing capacity was evaluated: vibrotactile threshold, amplitude discriminative capacity, and adaptation. It was found that warming the skin made a significant improvement on a subject's ability to detect a vibrotactile stimulus, and although the subjects' capacities for discriminating between two amplitudes of vibrotactile stimulation did not change with skin heating, the impact that adapting or conditioning stimulation normally had on amplitude discrimination capacity was significantly attenuated by the change in temperature. These results suggested that although the improvements in tactile sensitivity that were observed could have been a result of enhanced peripheral activity, the changes in measures that reflect a decrease in the sensitization to repetitive stimulation are most likely centrally mediated. The authors speculate that these centrally mediated changes could be a reflection of a change in the balance of cortical excitation and inhibition. |
The localization of an axon growth inhibitory molecule Nogo and its receptor (NgR) was investigated in the mouse spinal cord during prenatal development of the commissural pathway. Using the antibody N18, an intense signal for Nogo was localized largely on radial glia processes that are immunoreactive to RC2 antibody during the major period of commissural axon growth and was gradually reduced towards the end of gestation. The glial processes ramified extensively in the ventral funiculus and resided within the interfascicular space between the longitudinally projecting axons. Axonal localization of Nogo was observed on the premidline segment of commissural axons and on axons in the dorsal and ventral funiculi, but only at the earliest stage of pathway development. Nogo signals were initially weak on the glial processes during the period of axon crossing in the floor plate but was elevated when the decussation is finished. NgR was expressed on the commissural axons; the expression pattern is spatially regulated, being low in the premidline and midline courses but is upregulated when the axons leave the floor plate. These expression patterns raise the possibilities that the glial-specific form of Nogo may be involved in the guidance of commissural axons by (i) preventing recrossing of axons across the midline through an upregulation of axonal NgR and (ii) partitioning axons in the ventral funiculus into longitudinal fascicles. |
The aim of this study was to quantitatively investigate the chronic ethanol-induced cerebral metabolic changes in various regions of the rat brain, using the proton high resolution magic angle spinning spectroscopy technique. The rats were divided into two groups (control group: N=11, ethanol-treated group: N=11) and fed with the liquid diets for 10 weeks. In each week, the mean intake volumes of liquid diet were measured. The brain tissues, including cerebellum (Cere), frontal cortex (FC), hippocampus (Hip), occipital cortex (OC) and thalamus (Thal), were harvested immediately after the end of experiments. The ex vivo proton spectra for the five brain regions were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence at 500-MHz NMR spectrometer. All of the spectra were processed using the LCModel software, with simulated basis-set file, and the metabolite levels were referenced to total creatine. In the ethanol liquid diet group, there were significant increases in the metabolites ratio levels, as compared to control (Cere: alanine, glutathione, and N-acetlyaspartate; FC: phosphocholine and taurine; Hip: alanine, glutamine, and N-acetylaspartate; OC: glutamine; Thal: alanine, γ-aminobutyric acid, glutamate, glycerophosphocholine, phosphocholine, taurine, and free choline). However, in the ethanol liquid diet group, the myo-inositol levels of the OC were significantly lower. The present study demonstrates how chronic ethanol consumption affects cerebral metabolites in the chronic ethanol-treated rat. Therefore, this result could be useful to pursue clinical applications for quantitative diagnosis in human alcoholism. |
We have shown previously that intracerebroventricular (icv) injection of naloxone (a non-selective opioid receptor antagonist) or naloxonazine (a selective μ1-opioid receptor antagonist) at the maintenance phase of hibernation arouses Syrian hamsters from hibernation. This study was designed to clarify the role of β-endorphin (an endogenous μ-opioid receptor ligand) on regulation of body temperature (T(b)) during the maintenance phase of hibernation. The number of c-Fos-positive cells and β-endorphin-like immunoreactivity increased in the arcuate nucleus (ARC) after hibernation onset. In contrast, endomorphin-1 (an endogenous μ-opioid receptor ligand)-like immunoreactivity observed on the anterior hypothalamus decreased after hibernation onset. In addition, hibernation was interrupted by icv injection of anti-β-endorphin antiserum at the maintenance phase of hibernation. The mRNA expression level of proopiomelanocortin (a precursor of β-endorphin) on ARC did not change throughout the hibernation phase. However, the mRNA expression level of prohormone convertase-1 increased after hibernation onset. [D-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO, a selective μ-opioid receptor agonist) microinjection into the dorsomedial hypothalamus (DMH) elicited the most marked T(b) decrease than other sites such as the preoptic area (PO), anterior hypothalamus (AH), lateral hypothalamus (LH), ventromedial hypothalamus and posterior hypothalamus (PH). However, microinjected DAMGO into the medial septum indicated negligible changes in T(b). These results suggest that β-endorphin which synthesizes in ARC neurons regulates T(b) during the maintenance phase of hibernation by activating μ-opioid receptors in PO, AH, VMH, DMH and PH. |
Although anatomical data indicates that the caudal ventrolateral medulla (CVLM) projects directly to the subfornical organ (SFO), little is known about the afferent information relayed through the CVLM to SFO. Experiments were done in the anesthetized rat to investigate whether CVLM neurons mediate baroreceptor afferent information to SFO and whether this afferent information alters the response of SFO neurons to systemic injections of angiotensin II (ANG II). Extracellular single unit recordings were made from 78 spontaneously discharging single units in SFO. Of these, 32 (41%) responded to microinjection of L-glutamate (L-Glu; 0.25M; 10nl) into CVLM (27/32 were inhibited and 5/32 were excited). All 32 units also were excited by systemic injections of ANG II (250ng/0.1ml, ia). However, only those units inhibited by CVLM (n=27) were found to be inhibited by the reflex activation of baroreceptors following systemic injections of phenylephrine (2μg/kg, iv). Activation of CVLM or arterial baroreceptors in conjunction with ANG II resulted in an attenuation of the SFO unit's response to ANG II. Finally, microinjections (100nl) of the synaptic blocker CoCl(2) or the non-specific glutamate receptor antagonist kynurenic acid into CVLM attenuated (10/13 units tested) the SFO neuron's response to activation of baroreceptors, but not the unit's response evoked by systemic ANG II. Taken together, these data suggest that baroreceptor afferent information relayed through CVLM functions to modulate of the activity of neurons within SFO to extracellular signals of body fluid balance. |
[11C]CUMI-101 is the first selective serotonin receptor (5-HT1AR) partial agonist radiotracer for positron emission tomography (PET) tested in vivo in nonhuman primates and humans. We evaluated specific binding of [3H]CUMI-101 by quantitative autoradiography studies in postmortem baboon and human brain sections using the 5-HT1AR antagonist WAY-100635 as a displacer. The regional and laminar distributions of [3H]CUMI-101 binding in baboon and human brain sections matched the known distribution of [3H]8-OH-DPAT and [3H]WAY-100635. Prazosin did not measurably displace [3H]CUMI-101 binding in baboon or human brain sections, thereby ruling out [3H]CUMI-101 binding to α1-adrenergic receptors. This study demonstrates that [11C]CUMI-101 is a selective 5-HT1AR ligand for in vivo and in vitro studies in baboon and human brain. |
Prosaposin (also known as SGP-1) is an intriguing multifunctional protein that plays roles both intracellularly, as a regulator of lysosomal enzyme function, and extracellularly, as a secreted factor with neuroprotective and glioprotective effects. Following secretion, prosaposin can undergo endocytosis via an interaction with the low-density lipoprotein-related receptor 1 (LRP1). The ability of secreted prosaposin to promote protective effects in the nervous system is known to involve activation of G proteins, and the orphan G protein-coupled receptors GPR37 and GPR37L1 have recently been shown to mediate signaling induced by both prosaposin and a fragment of prosaposin known as prosaptide. In this review, we describe recent advances in our understanding of prosaposin, its receptors and their importance in the nervous system. |
Locus coeruleus (LC) nucleus is involved in noradrenergic descending pain modulation. LC receives dense orexinergic projections from the lateral hypothalamus. Orexin-A and -B are hypothalamic peptides which modulate a variety of brain functions via orexin type-1 (OX1) and orexin type-2 (OX2) receptors. Previous studies have shown that activation of OX1 receptors induces endocannabinoid synthesis and alters synaptic neurotransmission by retrograde signaling via affecting cannabinoid type-1 (CB1) receptors. In the present study the interaction of orexin-A and endocannabinoids was examined at the LC level in a rat model of inflammatory pain. Pain was induced by formalin (2%) injection into the hind paw. Intra-LC microinjection of orexin-A decreased the nociception score during both phases of formalin test. Furthermore, intra-LC microinjection of either SB-334867 (OX1 receptor antagonist) or AM251 (CB1 receptor antagonist) increased flinches and also the nociception score during phase 1, 2 and the inter-phase of formalin test. The analgesic effect of orexin-A was diminished by prior intra-LC microinjection of either SB-334867 or AM251. This data show that, activation of OX1 receptors in the LC can induce analgesia and also the blockade of OX1 or CB1 receptors is associated with hyperalgesia during formalin test. Our findings also suggest that CB1 receptors may modulate the analgesic effect of orexin-A. These results outline a new mechanism by which orexin-A modulates the nociceptive processing in the LC nucleus. |
Epidemiological studies indicate that light-moderate alcohol (ethanol) consumers tend to have reduced risks of cognitive impairment and progression to dementia during aging. Exploring possible mechanisms, we previously found that moderate ethanol preconditioning (MEP, 20-30mM) of rat brain cultures for several days instigated neuroprotection against β-amyloid peptides. Our biochemical evidence implicated the NMDA receptor (NMDAR) as a potential neuroprotective "sensor", specifically via synaptic NMDAR signaling. It remains unclear how ethanol modulates the receptor and its downstream targets to engender neuroprotection. Here we confirm with deconvolution microscopy that MEP of rat mixed cerebellar cultures robustly increases synaptic NMDAR localization. Phospho-activation of the non-receptor tyrosine kinases Src and Pyk2, known to be linked to synaptic NMDAR, is also demonstrated. Additionally, the preconditioning enhances levels of an antioxidant protein, peroxiredoxin 2 (Prx2), reported to be downstream of synaptic NMDAR signaling, and NMDAR antagonism with memantine (earlier found to abrogate MEP neuroprotection) blocks the Prx2 elevations. To further link Prx2 with antioxidant-based neuroprotection, we circumvented the ethanol preconditioning-NMDAR pathway by pharmacologically increasing Prx2 with the naturally-occurring cruciferous compound, 3H-1,2-dithiole-3-thione (D3T). Thus, D3T pretreatment elevated Prx2 expression to a similar extent as MEP, while concomitantly preventing β-amyloid neurotoxicity; D3T also protected the cultures from hydrogen peroxide toxicity. The findings support a mechanism that couples synaptic NMDAR signaling, Prx2 expression and augmented antioxidant defenses in ethanol preconditioning-induced neuroprotection. That this mechanism can be emulated by a cruciferous vegetable constituent suggests that such naturally-occurring "neutraceuticals" may be useful in therapy for oxidative stress-related dementias. |
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by loss of memory and cognitive abilities. In AD, amyloid β (Aβ) protein aggregates in the brain of patients, forming amyloid plaques. Aβ plaques are known to be surrounded by activated microglial cells. Serum amyloid A (SAA) is elevated from several hundred to 1000-fold as part of the immune response against various injuries, including trauma, infection, and inflammation. Additionally, continuous elevation of SAA is related to the development of amyloidosis. This study was designed to identify the relationship between SAA1 and AD using liver specific SAA1 overexpressing mice (TG), because SAA1 is expressed in the liver during the acute phase. We detected exogenous SAA1 expression in the brain of TG mice. This result implies that liver-derived SAA1 migrates to the brain tissues. Thus, we confirmed that the blood brain barrier (BBB) functioned normally using Evans-blue staining and CARS. Furthermore, our results show an increase in the accumulation of the 87kDa form of Aβ in TG mice compared to wild type mice (WT). Additionally, the number of microglial cells and levels of pro-inflammatory cytokines were increased. Next, we investigated the relationship between SAA1 and depression by performing social interaction tests. The results showed that TG mice have a tendency to avoid stranger mice and an impaired social recognition. In conclusion, the SAA1 TG mouse model is a valuable model to study depression. |
Uridine is a potential endogenous neuromodulator studied for several decades for its antiepileptic effect, but the results were controversial. One remarkable feature of uridine is its regulatory action on the dopaminergic pathways. In this study, the changes in uridine and dopamine (DA) release were examined in the mouse corpus striatum after pilocarpine (PC) intraperitoneal injection. Then, the effect of uridine pre-treatment on DA release and expression of dopamine receptor (DR) was determined. The results revealed an increased uridine release initially, followed by a downward trend after an injection of 400-mg/kg PC. However, the DA release continuous increased significantly. The expression of dopamine receptor-1 (D1R) increased in a dose-dependent manner while that of dopamine receptor-2 (D2R) decreased significantly. Prophylactic administration of uridine significantly relieved the high-frequency and high-amplitude expression induced by PC as well as dose-dependently reversed the PC-induced changes in DA and DRs levels. These findings suggested that uridine produced an antiepileptic effect, which might have been mediated in part by interfering with the dopaminergic system. |
Medial frontal activity in the EEG is enhanced following negative feedback and varies in relation to dimensions of impulsivity. In 22 undergraduate students (M<sub>age</sub> = 18.92 years, range 18-22 years), we employed a probabilistic negative reinforcement learning paradigm in which choices to avoid were followed by cues indicating successful or unsuccessful avoidance of an impending aversive noise. Our results showed that medial frontal theta power was enhanced following a cue that signaled avoidance was unsuccessful. In addition, self-reported lack of perseverance, a dimension of impulsivity characterized by an inability to maintain focus and determination during a challenging task, was negatively correlated with medial frontal theta elicited to an unsuccessful avoidance cue. We also observed robust differences in alpha attenuation and beta modulation following unsuccessful avoidance cue presentation. To our knowledge, this is the first study in humans to show a functional relation between medial frontal theta modulation and avoidance success. We discuss our findings in the context of frontal theta and self-regulation, negative reinforcement, and anxiety. |
We emulated instances of open traumatic brain injuries (TBI) in a maritime disaster. New Zealand rabbit animal models were used to evaluate the pathophysiological changes in open TBI with and without the influence of artificial seawater. New Zealand rabbits were randomly divided into 3 groups. Control group consisted of only normal animals. Animals in TBI and TBI + Seawater groups underwent craniotomy with dura mater incised and brain tissue exposed to free-fall impact. Afterward, only TBI + Seawater group received on-site artificial seawater infusion. Brain water content (BWC) and permeability of blood-brain barrier (BBB) were assessed. Reactive oxygen species levels were measured. Western blotting and immunofluorescence were employed to detect: apoptosis-related factors Caspase-3, Bax and Bcl-2; angiogenesis-related factors CD31 and CD34; astrogliosis-related factor glial fibrillary acidic protein (GFAP); potential neuron injury indicator neuron-specific enolase (NSE). Hematoxylin & eosin, Masson-trichrome and Nissl stainings were performed for pathological observations. Comparing to Control group, TBI group manifested abnormal neuronal morphology; increased BWC; compromised BBB integrity; increased ROS, Bax, CD31, CD34, Caspase-3 and GFAP expressions; decreased Bcl-2 and NSE expression. Seawater immersion caused all changes, except BWC, to become more significant. Seawater immersion worsens the damage inflicted to brain tissue by open TBI. It aggravates hypoxia in brain tissue, upregulates ROS expression, increases neuron sensitivity to apoptosis-inducing factors, and promotes angiogenesis as well as astrogliosis. |
Intracerebral hemorrhage (ICH) is a subtype of stroke that causes major motor impairments. Brain-derived neurotrophic factor (BDNF) is known to have important roles in neuroplasticity and beneficially contributes to stroke recovery. This study aimed to characterize BDNF expression in the motor cortex after ICH and investigate the relationship between cortical BDNF expression and behavioral outcomes using an ICH rat model. Wistar rats were divided into two groups: a SHAM group (n = 7) and an ICH group (n = 8). ICH was induced by the injection of collagenase into the left striatum near the internal capsule. For behavioral assessments, the cylinder test and open field test were performed before surgery and 3 days, 1 week, 2 weeks, and 4 weeks after surgery. Following the behavioral assessments at 4 weeks, BDNF expression in the ipsilateral and contralateral motor cortex was assayed using RT-PCR and ELISA methods. There was no significant difference in either cortical BDNF mRNA or protein expression levels between the SHAM and ICH groups. However, the asymmetry index of BDNF mRNA expression between the ipsilateral and contralateral hemispheres shifted to the ipsilateral hemisphere after ICH. Furthermore, the ipsilateral cortical BDNF mRNA expression level positively correlated with motor function in the affected forelimb after ICH. This study describes for the first time that cortical BDNF mRNA expression is related to post-ICH motor impairment. These results highlight the importance of assessing the interhemispheric laterality of BDNF expression and could help develop novel treatment strategies for BDNF-dependent recovery after ICH. |
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