Staff member

(Phospho)proteomics of old-aged subjects without cognitive or behavioral symptoms, and without AD-neuropathological changes and lacking any other neurodegenerative alteration will increase understanding about the physiological state of human brain aging without associate neurological deficits and neuropathological lesions.(Phospho)proteomics using conventional label-free- and SWATH-MS (Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) has been assessed in the frontal cortex (FC) of individuals without NFTs, senile plaques (SPs) and age-related co-morbidities classified by age (years) in four groups; group 1 (young, 30-44); group 2 (middle-aged: MA, 45-52); group 3 (early-elderly, 64-70); and group 4 (late-elderly, 75-85).Protein levels and deregulated protein phosphorylation linked to similar biological terms/functions, but involving different individual proteins, are found in FC with age. The modified expression occurs in cytoskeleton proteins, membranes, synapses, vesicles, myelin, membrane transport and ion channels, DNA and RNA metabolism, ubiquitin-proteasome-system (UPS), kinases and phosphatases, fatty acid metabolism, and mitochondria. Dysregulated phosphoproteins are associated with the cytoskeleton, including microfilaments, actin-binding proteins, intermediate filaments of neurons and glial cells, and microtubules; membrane proteins, synapses, and dense core vesicles; kinases and phosphatases; proteins linked to DNA and RNA; members of the UPS; GTPase regulation; inflammation; and lipid metabolism. Noteworthy, protein levels of large clusters of hierarchically-related protein expression levels are stable until 70. However, protein levels of components of cell membranes, vesicles and synapses, RNA modulation, and cellular structures (including tau and tubulin filaments) are markedly altered from the age of 75. Similarly, marked modifications occur in the larger phosphoprotein clusters involving cytoskeleton and neuronal structures, membrane stabilization, and kinase regulation in the late elderly.Present findings may increase understanding of human brain proteostasis modifications in the elderly in the subpopulation of individuals not having AD neuropathological change and any other neurodegenerative change in any telencephalon region.

JTD Keywords: (phospho)proteomics, cortex, cytoskeleton, hippocampus, kinases, membranes, mitochondria, mitochondrial-function, pathological process, phosphoproteome analysis, phosphorylation, proteome, quantitative proteomics, synapsis, tau-protein, therapeutic target, (phospho)proteomics, Aged, Alzheimer disease, Brain, Brain aging, Cytoskeleton, Humans, Kinases, Membranes, Middle aged, Mitochondria, Nervous system diseases, Neurodegenerative diseases, Neurons, Phosphoric monoester hydrolases, Proteome, Synapsis, Tau proteins

The hippocampus of cases with neurofibrillary tangles (NFT) pathology classified as stages I–II, III–IV, and V–VI without comorbidities, and middle-aged (MA) individuals with no NFT pathology, were examined to learn about the composition of granulovacuolar degeneration (GVD). Our results confirm the presence of CK1-?, p38-P Thr180/Tyr182, SAPK/JNK-P Thr183/Thr185, GSK-3?/?-P Tyr279/Tyr216, and GSK-3? Ser9 in the cytoplasmic granules in a subset of neurons of the CA1 and CA2 subfields of the hippocampus. Also, we identify the presence of PKA ?/?-P Thr197, SRC-P Tyr416, PAK1-P Ser199/Ser204, CAMK2A-P Tyr197, and PKCG-P Thr655 in cytoplasmic granules in cases with NFT pathology, but not in MA cases. Our results also confirm the presence of ?-catenin-P Ser45/Thr41, IRE?-P Ser274, eIF2?-P Ser51, TDP-43-P Ser403-404 (but absent TDP-43), and ubiquitin in cytoplasmic granules. Other components of the cytoplasmic granules are MAP2-P Thr1620/1623, MAP1B-P Thr1265, ADD1-P Ser726, and ADD1/ADD1-P Ser726/Ser713, in addition to several tau species including 3Rtau, 4Rtau, and tau-P Ser262. The analysis of GVD at progressive stages of NFT pathology reveals the early appearance of phosphorylated kinases and proteins in cytoplasmic granules at stages I–II, before the appearance of pre-tangles and NFTs. Most of these granules are not surrounded by LAMP1-positive membranes. Markers of impaired ubiquitin-protesome system, abnormal reticulum stress response, and altered endocytic and autophagic pathways occur in a subpopulation of neurons containing cytoplasmic granules, and they appear later. These observations suggest early phosphorylation of kinases leading to their activation, and resulting in the abnormal phosphorylation of various substrates, including tau, as a main alteration at the first stages of GVD. © 2021 The Author(s)

JTD Keywords: alzheimer's disease, alzheimers association guidelines, alzheimer’s disease, brain aging, cyclin-dependent kinase-5, granulovacuolar degeneration, kinases, national institute, neuropathologic assessment, p38 kinase, progressive supranuclear palsy, protein phosphorylation, tau, tau pathology, up-regulation, upstream activator, Alzheimer disease, Alzheimer's disease, Alzheimer’s disease, Brain aging, Dna-binding proteins, Glycogen synthase kinase 3 beta, Glycogen-synthase kinase-3, Granulovacuolar degeneration, Hippocampus, Humans, Kinases, Middle aged, Nerve degeneration, Neurofibrillary tangles, Phosphorylation, Protein phosphorylation, Tau, Tau proteins, Ubiquitins

Transcriptomics and phosphoproteomics were carried out in the cerebral cortex of B6.Cg-Mapttm1(EGFP)Klt (tau knockout: tau-KO) and wild-type (WT) 12 month-old mice to learn about the effects of tau ablation. Compared with WT mice, tau-KO mice displayed reduced anxiety-like behavior and lower fear expression induced by aversive conditioning, whereas recognition memory remained unaltered. Cortical transcriptomic analysis revealed 69 downregulated and 105 upregulated genes in tau-KO mice, corresponding to synaptic structures, neuron cytoskeleton and transport, and extracellular matrix components. RT-qPCR validated increased mRNA levels of col6a4, gabrq, gad1, grm5, grip2, map2, rab8a, tubb3, wnt16, and an absence of map1a in tau-KO mice compared with WT mice. A few proteins were assessed with Western blotting to compare mRNA expression with corresponding protein levels. Map1a mRNA and protein levels decreased. However, β-tubulin III and GAD1 protein levels were reduced in tau-KO mice. Cortical phosphoproteomics revealed 121 hypophosphorylated and 98 hyperphosphorylated proteins in tau-KO mice. Deregulated phosphoproteins were categorized into cytoskeletal (n = 45) and membrane proteins, including proteins of the synapses and vesicles, myelin proteins, and proteins linked to membrane transport and ion channels (n = 84), proteins related to DNA and RNA metabolism (n = 36), proteins connected to the ubiquitin-proteasome system (UPS) (n = 7), proteins with kinase or phosphatase activity (n = 21), and 22 other proteins related to variegated pathways such as metabolic pathways, growth factors, or mitochondrial function or structure. The present observations reveal a complex altered brain transcriptome and phosphoproteome in tau-KO mice with only mild behavioral alterations.

JTD Keywords: computational platform, conformational-changes, cytoskeleton, disease, expression, isoforms, mechanisms, mice, phosphoproteomics, phosphorylation, synapse, tau-ko, tauopathies, transcriptomics, Animals, Cerebral cortex, Cytoskeleton, Grip2 protein, mouse, Intracellular signaling peptides and proteins, Mapt protein, mouse, Mice, Mice, knockout, Nerve tissue proteins, Neurons, Phosphoproteomics, Proteostasis, Rna, messenger, Synapse, Tau proteins, Tau-ko, Tau-protein, Transcriptomics

Heterozygous hTau mice were used for the study of tau seeding. These mice express the six human tau isoforms, with a high predominance of 3Rtau over 4Rtau. The following groups were assessed: (i) non-inoculated mice aged 9 months (n = 4); (ii) Alzheimer's Disease (AD)-inoculated mice (n = 4); (iii) Globular Glial Tauopathy (GGT)-inoculated mice (n = 4); (iv) Pick's disease (PiD)-inoculated mice (n = 4); (v) control-inoculated mice (n = 4); and (vi) inoculated with vehicle alone (n = 2). AD-inoculated mice showed AT8-immunoreactive neuronal pre-tangles, granular aggregates, and dots in the CA1 region of the hippocampus, dentate gyrus (DG), and hilus, and threads and dots in the ipsilateral corpus callosum. GGT-inoculated mice showed unique or multiple AT8-immunoreactive globular deposits in neurons, occasionally extended to the proximal dendrites. PiD-inoculated mice showed a few loose pre-tangles in the CA1 region, DG, and cerebral cortex near the injection site. Coiled bodies were formed in the corpus callosum in AD-inoculated mice, but GGT-inoculated mice lacked globular glial inclusions. Tau deposits in inoculated mice co-localized active kinases p38-P and SAPK/JNK-P, thus suggesting active phosphorylation of the host tau. Tau deposits were absent in hTau mice inoculated with control homogenates and vehicle alone. Deposits in AD-inoculated hTau mice contained 3Rtau and 4Rtau; those in GGT-inoculated mice were mainly stained with anti-4Rtau antibodies, but a small number of deposits contained 3Rtau. Deposits in PiD-inoculated mice were stained with anti-3Rtau antibodies, but rare neuronal, thread-like, and dot-like deposits showed 4Rtau immunoreactivity. These findings show that tau strains produce different patterns of active neuronal seeding, which also depend on the host tau. Unexpected 3Rtau and 4Rtau deposits after inoculation of homogenates from 4R and 3R tauopathies, respectively, suggests the regulation of exon 10 splicing of the host tau during the process of seeding, thus modulating the plasticity of the cytoskeleton.

JTD Keywords: alzheimer's disease (ad), alzheimers-disease, brain, corticobasal degeneration, globular glial tauopathy (ggt), htau, isoforms, pathological tau, pick's disease (pid), picks-disease, propagation, protein, seeding, tau splicing, tauopathy, Alzheimer disease, Alzheimer’s disease (ad), Animals, Brain, Globular glial tauopathy (ggt), Hippocampus, Htau, Humans, Mice, Mice, transgenic, Paired helical filaments, Pick disease of the brain, Pick’s disease (pid), Seeding, Tau, Tau proteins, Tau splicing, Tauopathies

Alzheimer’s disease (AD) and other tauopathies are common neurodegenerative diseases in older adults; in contrast, abnormal tau deposition in neurons and glial cells occurs only exceptionally in children. Sarkosyl-insoluble fractions from sporadic AD (sAD) containing paired helical filaments (PHFs) were inoculated unilaterally into the thalamus in newborn and three-month-old wild-type C57BL/6 mice, which were killed at different intervals from 24 h to six months after inoculation. Tau-positive cells were scanty and practically disappeared at three months in mice inoculated at the age of a newborn. In contrast, large numbers of tau-positive cells, including neurons and oligodendrocytes, were found in the thalamus of mice inoculated at three months and killed at the ages of six months and nine months. Mice inoculated at the age of newborn and re-inoculated at the age of three months showed similar numbers and distribution of positive cells in the thalamus at six months and nine months. This study shows that (a) differences in tau seeding between newborn and young adults may be related to the ratios between 3Rtau and 4Rtau, and the shift to 4Rtau predominance in adults, together with the immaturity of connections in newborn mice, and (b) intracerebral inoculation of sAD PHFs in newborn mice does not protect from tau seeding following intracerebral inoculation of sAD PHFs in young/adult mice.

JTD Keywords: alzheimer's disease, alzheimer-disease, alzheimer’s disease, expression, mouse tau, neurofibrillary tangles, newborn, pathological tau, propagation, protein-tau, spread, tau seeding and spreading, thalamus, transgenic mice, Alzheimer disease, Alzheimer’s disease, Animals, Brain, Mice, Mice, inbred c57bl, Mice, transgenic, Neurofibrillary tangles, Newborn, Paired helical filaments, Tau proteins, Tau seeding and spreading, Tauopathies, Thalamus

Several studies have demonstrated the different characteristics of tau seeding and spreading following intracerebral inoculation in murine models of tau-enriched fractions of brain homogenates from AD and other tauopathies. The present study is centered on the importance of host tau in tau seeding and the molecular changes associated with the transformation of host tau into abnormal tau. The brains of three adult murine genotypes expressing different forms of tau—WT (murine 4Rtau), hTau (homozygous transgenic mice knock-out for murine tau protein and heterozygous expressing human forms of 3Rtau and 4Rtau proteins), and mtWT (homozygous transgenic mice knock-out for murine tau protein)—were analyzed following unilateral hippocampal inoculation of sarkosyl-insoluble tau fractions from the same AD and control cases. The present study reveals that (a) host tau is mandatory for tau seeding and spreading following tau inoculation from sarkosyl-insoluble fractions obtained from AD brains; (b) tau seeding does not occur following intracerebral inoculation of sarkosyl-insoluble fractions from controls; (c) tau seeding and spreading are characterized by variable genotype-dependent tau phosphorylation and tau nitration, MAP2 phosphorylation, and variable activation of kinases that co-localize with abnormal tau deposits; (d) transformation of host tau into abnormal tau is an active process associated with the activation of specific kinases; (e) tau seeding is accompanied by modifications in tau splicing, resulting in the expression of new 3Rtau and 4Rtau isoforms, thus indicating that inoculated tau seeds have the capacity to model exon 10 splicing of the host mapt or MAPT with a genotype-dependent pattern; (e) selective regional and cellular vulnerabilities, and different molecular compositions of the deposits, are dependent on the host tau of mice injected with identical AD tau inocula.

JTD Keywords: 3rtau and 4rtau, alzheimer's disease, alzheimer’s disease, brains, granulovacuolar degeneration, host tau, htau, intranuclear distribution, messenger-rna, pathological tau, propagation, protein-kinases, seeding and spreading, tauopathies, transmission, 3rtau and 4rtau, Alzheimer disease, Alzheimers-disease, Alzheimer’s disease, Animals, Biomarkers, Brain, Disease models, animal, Disease susceptibility, Fluorescent antibody technique, Genotype, Hippocampus, Host tau, Htau, Humans, Immunohistochemistry, Mice, Mice, knockout, Mice, transgenic, Mutation, Neurons, Seeding and spreading, Tau proteins, Tauopathies

Globular glial tauopathy (GGT) is a progressive neurodegenerative disease involving the grey matter and white matter (WM) and characterized by neuronal deposition of hyper-phosphorylated, abnormally conformed, truncated, oligomeric 4Rtau in neurons and in glial cells forming typical globular astrocyte and oligodendrocyte inclusions (GAIs and GOIs, respectively) and coiled bodies. Present studies centre on four genetic GGT cases from two unrelated families bearing the P301T mutation in MAPT and one case of sporadic GGT (sGGT) and one case of GGT linked to MAPT K317M mutation, for comparative purposes. Clinical and neuropathological manifestations and biochemical profiles of phospho-tau are subjected to individual variations in patients carrying the same mutation, even in carriers of the same family, independently of the age of onset, gender, and duration of the disease. Immunohistochemistry, western blotting, transcriptomic, proteomics and phosphoproteomics, and intra-cerebral inoculation of brain homogenates to wild-type (WT) mice were the methods employed. In GGT cases linked to MAPT P301T mutation, astrocyte markers GFAP, ALDH1L1, YKL40 mRNA and protein, GJA1 mRNA, and AQ4 protein are significantly increased; glutamate transporter GLT1 (EAAT2) and glucose transporter (SLC2A1) decreased; mitochondrial pyruvate carrier 1 (MPC1) increased, and mitochondrial uncoupling protein 5 (UCP5) almost absent in GAIs in frontal cortex (FC). Expression of oligodendrocyte markers OLIG1 and OLIG2mRNA, and myelin-related genes MBP, PLP1, CNP, MAG, MAL, MOG, and MOBP are significantly decreased in WM; CNPase, PLP1, and MBP antibodies reveal reduction and disruption of myelinated fibres; and SMI31 antibodies mark axonal damage in the WM. Altered expression of AQ4, GLUC-t, and GLT-1 is also observed in sGGT and in GGT linked to MAPT K317M mutation. These alterations point to primary astrogliopathy and oligodendrogliopathy in GGT. In addition, GGT linked to MAPT P301T mutation proteotypes unveil a proteostatic imbalance due to widespread (phospho)proteomic dearrangement in the FC and WM, triggering a disruption of neuron projection morphogenesis and synaptic transmission. Identification of hyper-phosphorylation of variegated proteins calls into question the concept of phospho-tau-only alteration in the pathogenesis of GGT. Finally, unilateral inoculation of sarkosyl-insoluble fractions of GGT homogenates from GGT linked to MAPT P301T, sGGT, and GGT linked to MAPT K317M mutation in the hippocampus, corpus callosum, or caudate/putamen in wild-type mice produces seeding, and time- and region-dependent spreading of phosphorylated, non-oligomeric, and non-truncated 4Rtau and 3Rtau, without GAIs and GOIs but only of coiled bodies. These experiments prove that host tau strains are important in the modulation of cellular vulnerability and phenotypes of phospho-tau aggregates.

JTD Keywords: Globular glial tauopathy, Tau, Astrogliopathy, Oligodendrogliopathy, Phosphoproteome, Seeding and spreading

Human tau seeding and spreading occur following intracerebral inoculation of brain homogenates obtained from tauopathies in transgenic mice expressing natural or mutant tau, and in wild-type (WT) mice. The present study was geared to learning about the patterns of tau seeding, the cells involved and the characteristics of tau following intracerebral inoculation of homogenates from primary age-related tauopathy (PART: neuronal 4Rtau and 3Rtau), aging-related tau astrogliopathy (ARTAG: astrocytic 4Rtau) and globular glial tauopathy (GGT: 4Rtau with neuronal deposits and specific tau inclusions in astrocytes and oligodendrocytes). For this purpose, young and adult WT mice were inoculated unilaterally in the hippocampus or in the lateral corpus callosum with sarkosyl-insoluble fractions from PART, ARTAG and GGT cases, and were killed at variable periods of three to seven months. Brains were processed for immunohistochemistry in paraffin sections. Tau seeding occurred in the ipsilateral hippocampus and corpus callosum and spread to the septal nuclei, periventricular hypothalamus and contralateral corpus callosum, respectively. Tau deposits were mainly found in neurons, oligodendrocytes and threads; the deposits were diffuse or granular, composed of phosphorylated tau, tau with abnormal conformation and 3Rtau and 4Rtau independently of the type of tauopathy. Truncated tau at the aspartic acid 421 and ubiquitination were absent. Tau deposits had the characteristics of pre-tangles. A percentage of intracellular tau deposits co-localized with active (phosphorylated) tau kinases p38 and ERK 1/2. Present study shows that seeding and spreading of human tau into the brain of WT mice involves neurons and glial cells, mainly oligodendrocytes, thereby supporting the idea of a primary role of oligodendrogliopathy, together with neuronopathy, in the progression of tauopathies. In addition, it suggests that human tau inoculation modifies murine tau metabolism with the production and deposition of 3Rtau and 4Rtau, and by activation of specific tau kinases in affected cells.

JTD Keywords: Aging-related tau astrogliopathy, Globular glial tauopathy, Primary age-related tauopathy, Seeding, Spreading, Tau, Tauopathies

Introduction: Human tau seeding and spreading occur following intracerebral inoculation into different gray matter regions of brain homogenates obtained from tauopathies in transgenic mice expressing wild or mutant tau, and in wild-type (WT) mice. However, little is known about tau propagation following inoculation in the white matter. Objectives: The present study is geared to learning about the patterns of tau seeding and cells involved following unilateral inoculation in the corpus callosum of homogenates from sporadic Alzheimer's disease (AD), primary age-related tauopathy (PART: neuronal 4Rtau and 3Rtau), pure aging-related tau astrogliopathy (ARTAG: astroglial 4Rtau with thorn-shaped astrocytes TSAs), globular glial tauopathy (GGT: 4Rtau with neuronal tau and specific tau inclusions in astrocytes and oligodendrocytes, GAIs and GOIs, respectively), progressive supranuclear palsy (PSP: 4Rtau with neuronal inclusions, tufted astrocytes and coiled bodies), Pick's disease (PiD: 3Rtau with characteristic Pick bodies in neurons and tau containing fibrillar astrocytes), and frontotemporal lobar degeneration linked to P301L mutation (FTLD-P301L: 4Rtau familial tauopathy). Methods: Adult WT mice were inoculated unilaterally in the lateral corpus callosum with sarkosyl-insoluble fractions or with sarkosyl-soluble fractions from the mentioned tauopathies; mice were killed from 4 to 7 months after inoculation. Brains were fixed in paraformaldehyde, embedded in paraffin and processed for immunohistochemistry. Results: Tau seeding occurred in the ipsilateral corpus callosum and was also detected in the contralateral corpus callosum. Phospho-tau deposits were found in oligodendrocytes similar to coiled bodies and in threads. Moreover, tau deposits co-localized with active (phosphorylated) tau kinases p38 and ERK 1/2, suggesting active tau phosphorylation of murine tau. TSAs, GAIs, GOIs, tufted astrocytes, and tau-containing fibrillar astrocytes were not seen in any case. Tau deposits were often associated with slight myelin disruption and the presence of small PLP1-immunoreactive globules and dots in the ipsilateral corpus callosum 6 months after inoculation of sarkosyl-insoluble fractions from every tauopathy. Conclusions: Seeding and spreading of human tau in the corpus callosum of WT mice occurs in oligodendrocytes, thereby supporting the idea of a role of oligodendrogliopathy in tau seeding and spreading in the white matter in tauopathies. Slight differences in the predominance of threads or oligodendroglial deposits suggest disease differences in the capacity of tau seeding and spreading among tauopathies.

JTD Keywords: AD, ARTAG, GGT, PiD, Seeding and spreading, Tau, Tauopathies

Human tau seeding and spreading occur following intracerebral inoculation of brain homogenates obtained from tauopathies in transgenic mice expressing natural or mutant tau, and in wild-type (WT) mice. The present study was geared to learning about the patterns of tau seeding, the cells involved and the characteristics of tau following intracerebral inoculation of homogenates from primary age-related tauopathy (PART: neuronal 4Rtau and 3Rtau), aging-related tau astrogliopathy (ARTAG: astrocytic 4Rtau) and globular glial tauopathy (GGT: 4Rtau with neuronal deposits and specific tau inclusions in astrocytes and oligodendrocytes). For this purpose, young and adult WT mice were inoculated unilaterally in the hippocampus or in the lateral corpus callosum with sarkosyl-insoluble fractions from PART, ARTAG and GGT cases, and were killed at variable periods of three to seven months. Brains were processed for immunohistochemistry in paraffin sections. Tau seeding occurred in the ipsilateral hippocampus and corpus callosum and spread to the septal nuclei, periventricular hypothalamus and contralateral corpus callosum, respectively. Tau deposits were mainly found in neurons, oligodendrocytes and threads; the deposits were diffuse or granular, composed of phosphorylated tau, tau with abnormal conformation and 3Rtau and 4Rtau independently of the type of tauopathy. Truncated tau at the aspartic acid 421 and ubiquitination were absent. Tau deposits had the characteristics of pre-tangles. A percentage of intracellular tau deposits co-localized with active (phosphorylated) tau kinases p38 and ERK 1/2. Present study shows that seeding and spreading of human tau into the brain of WT mice involves neurons and glial cells, mainly oligodendrocytes, thereby supporting the idea of a primary role of oligodendrogliopathy, together with neuronopathy, in the progression of tauopathies. In addition, it suggests that human tau inoculation modifies murine tau metabolism with the production and deposition of 3Rtau and 4Rtau, and by activation of specific tau kinases in affected cells.

JTD Keywords: Aging-related tau astrogliopathy, Globular glial tauopathy, Primary age-related tauopathy, Seeding, Spreading, Tau, Tauopathies

The expression of subunits of mitochondrial respiratory complexes and components of the protein synthesis machinery from the nucleolus to the ribosome was analyzed in the mediodorsal thalamus in seven cases of Fatal Familial Insomnia (FFI) compared with age-matched controls. NDUFB8 (complex I subunit), SDHB (complex II subunit), UQCRC2 (complex III subunit), COX2 (complex IV subunit) and ATP50 (complex V subunit) expression levels, as revealed by western blotting, were reduced in FFI. Voltage-dependent anion channel (VDAC) and ATP5H were also reduced due to the marked depopulation of neurons. In contrast, a marked increase in superoxide dismutase 2 (SOD2) was found in reactive astrocytes thus suggesting that astrocytes are key factors in oxidative stress responses. The histone-binding chaperones nucleolin and nucleoplasmin 3, and histone H3 di-methylated K9 were markedly reduced together with a decrease in the expression of protein transcription elongation factor eEF1A. These findings show severe impairment in the expression of crucial components of mitochondrial function and protein synthesis in parallel with neuron loss in mediodorsal thalamus at terminal stages of FFI. Therapeutic measures must be taken long before the appearance of clinical symptoms to prevent the devastating effects of FFI.

JTD Keywords: Fatal familial insomnia, Mitochondria, Protein synthesis, Mitochondrial respiratory chain, Nucleolus, Ribosome

Objectives: The goal of this study was to assess mitochondrial function, energy, and purine metabolism, protein synthesis machinery from the nucleolus to the ribosome, inflammation, and expression of newly identified ectopic olfactory receptors (ORs) and taste receptors (TASRs) in the frontal cortex of typical cases of dementia with Lewy bodies (DLB) and cases with rapid clinical course (rpDLB: 2 years or less) compared with middle-aged non-affected individuals, in order to learn about the biochemical abnormalities underlying Lewy body pathology. Methods: Real-time quantitative PCR, mitochondrial enzymatic assays, and analysis of β-amyloid, tau, and synuclein species were used. Results: The main alterations in DLB and rpDLB, which are more marked in the rapidly progressive forms, include (i) deregulated expression of several mRNAs and proteins of mitochondrial subunits, and reduced activity of complexes I, II, III, and IV of the mitochondrial respiratory chain; (ii) reduced expression of selected molecules involved in energy metabolism and increased expression of enzymes involved in purine metabolism; (iii) abnormal expression of nucleolar proteins, rRNA18S, genes encoding ribosomal proteins, and initiation factors of the transcription at the ribosome; (iv) discrete inflammation; and (v) marked deregulation of brain ORs and TASRs, respectively. Severe mitochondrial dysfunction involving activity of four complexes, minimal inflammatory responses, and dramatic altered expression of ORs and TASRs discriminate DLB from Alzheimer’s disease. Altered solubility and aggregation of α-synuclein, increased β-amyloid bound to membranes, and absence of soluble tau oligomers are common in DLB and rpDLB. Low levels of soluble β-amyloid are found in DLB. However, increased soluble β-amyloid 1–40 and β-amyloid 1–42, and increased TNFα mRNA and protein expression, distinguish rpDLB. Conclusion: Molecular alterations in frontal cortex in DLB involve key biochemical pathways such as mitochondria and energy metabolism, protein synthesis, purine metabolism, among others and are accompanied by discrete innate inflammatory response.

JTD Keywords: Dementia with Lewy bodies, Alzheimer’s disease, α-synuclein, Mitochondria, Protein synthesis, Inflammation, β-amyloid, Olfactory receptors

Creutzfeldt-Jakob disease (cJD) is a heterogenic neurodegenerative disorder associated with abnormal posttranslational processing of cellular prion protein (PrPc). cJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrPsc (type 1:20/21 kDa and type 2:19 kDa). MM1 and VV2 are the most common sporadic cJD (scJD) subtypes. PrP mRNa expression levels in the frontal cortex and cerebellum are reduced in scJD in a form subtype-dependent. Total PrP protein levels and PrPsc levels in the frontal cortex and cerebellum accumulate differentially in scJD MM1 and scJD VV2 with no relation between PrPsc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the cSF, reduced PrPc, the only form present in this compartment, occurs in scJD MM1 and VV2. PrP mRNa expression is also reduced in the frontal cortex in advanced stages of alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrPc levels in brain varies from one disease to another. Reduced PrPc levels in cSF correlate with PrP mRNa expression in brain, which in turn reflects severity of degeneration in scJD.

JTD

The potential use of a micromachined thermopile based sensor device for analyzing natural gas is explored. The sensor consists of a thermally isolated hotplate which is heated by the application of a sequence of programmed voltages to an integrated heater. Once the hotplate reaches a stationary temperature, the thermopile provides a signal proportional to the hotplate temperature. These signals are processed in order to determine different natural gas properties. Sensor response is mainly dependent on the thermal conductivity of the surrounding gas at different temperatures. Seven predicted properties (normal density, Superior Heating Value, Wobbe index and the concentrations of methane, ethane, carbon dioxide and nitrogen) are calibrated against sensor signals by using multivariate regression, in particular Partial Least Squares. Experimental data have been used for calibration and validation. Results show property prediction capability with reasonable accuracy except for prediction of carbon dioxide concentration. A detailed uncertainty analysis is provided to better understand the metrological limits of the system. These results imply for the first time the possibility of designing unprecedented low-cost natural gas analyzers. The concept may be extended to other constrained gas mixtures (e.g. of a known number of components) to enable low-cost multicomponent gas analyzers.

JTD Keywords: Gas sensor, Natural gas, MEMS, Superior Heating Value, density, PLS

A hybrid microfluidic device that uses hydrodynamic forces to separate human plasma from blood cells has been designed and fabricated and the advantageous effects of temperature and flow rates are investigated in this paper. The blood separating device includes an inlet which is reduced by approximately 20 times to a small constrictor channel, which then opens out to a larger output channel with a small lateral channel for the collection of plasma. When tested the device separated plasma from whole blood using a wide range of flow rates, between 50 mu l min(-1) and 200 mu l min(-1), at the higher flow rates injected by hand and at temperatures ranging from 23 degrees C to 50 degrees C, the latter resulting in an increase in the cell-free layer of up to 250%. It was also tested continuously using between 5% and 40% erythrocytes in plasma and whole blood without blocking the channels or hemolysis of the cells. The mean percentage of plasma collected after separation was 3.47% from a sample of 1 ml. The percentage of cells removed from the plasma varied depending on the flow rate used, but at 37 degrees C ranged between 95.4 +/- 1% and 97.05 +/- 05% at 100 mu l min(-1) and 200 mu l min(-1), respectively. The change in temperature also had an effect on the number of cells removed from the plasma which was between 93.5 +/- 0.65% and 97.01 +/- 0.3% at 26.9 degrees C and 37 degrees C, respectively, using a flow rate of 100 mu l min(-1). Due to its ability to operate in a wide range of conditions, it is envisaged that this device can be used in in vitro 'lab on a chip' applications, as well as a hand-held point of care (POC) device.

JTD Keywords: On-a-chip, Cells, Viscosity, Membrane

An analysis of accuracy of time-constant spectrum extraction from thermal transients has been performed. Numerical calculations based on analytical models and finite element method simulations have been used in order to obtain the thermal transients. Simple geometries have been used such that analytical expressions for their time-constant spectrums are known. Results show that a large error in the time-constant spectrum is obtained for very small rms error ( 1 mK) in the thermal transient. The estimation problem is ill-conditioned. Moreover, the differential structure function shows a low accuracy identifying stacked structures. The initial part of the differential structure function shows numerical oscillations and the final part has an asymptotic behavior to infinity that has been identified as an artifact related to errors in the time-constant spectrum estimation. Peak identification from the differential structure function heavily depends on an accurate determination of the time-constant spectrum. The limited spectral resolution and dynamic range of the differential structure function are a direct consequence of the time-constant spectrum imprecision.

JTD Keywords: Finite element analysis, Spectral analysis

In the trend towards miniaturization, a detector module containing multiple IR sensor channels is being built and characterized. In its final form it contains thermopiles, narrow band filters and Fresnel lenses. An important feature of such module is the assembly by flip-chip of the IR filters on top of the thermopiles. The performance of the filter-thermopile ensemble has been assessed by physical simulation and experiments and it has been optimized by the use of an empirically validated model. It has been found that integration of filters (or lenses) too close to the IR detector may lead to degraded performance due to thermal coupling. The impact and extent of this degradation has been thoroughly explored, being the main parameter the distance between the IR sensor and the filter. To avoid such detrimental effects a possibility is to set the device in vacuum conditions, obtaining an improved output response and avoiding the influence of the filters. Another way is to increase the solder joint height. Beyond a certain height, the filter is considered to be isolated from the thermopile.

JTD Keywords: Assembly, Infrared sensor, Infrared filter, Fresnel lenses, FEM simulation, Optimization

Natural gas may show significant changes in its chemical composition depending on its origin. Typically, natural gas analysis is carried out using process gas chromatography. However, other methods based on the evaluation of physical properties have recently been reported. Thermal conductivity sensors are currently used in the analysis of binary mixtures of dissimilar gases. In contrast, natural gas is a complex mixture of mainly hydrocarbons, plus other residual gases as carbon dioxide and nitrogen. In this work, the response of a micromachined sensor integrating a heater and a thermopile is studied, regarding its potential use for natural gas analysis. A finite element thermal model of the device is described, and thermal operation simulations as well as a preliminary sensitivity analysis are reported. Experimental data has been collected and compared with simulated data, showing very good agreement. Results show that small variations in the gas mixture composition can be clearly detected. The sensor appears as a good candidate to be included in low-cost natural gas property analysis and quality control systems.

JTD Keywords: Natural gas, Thermopile, MEMS, Thermal conductivity, Modeling, FEM simulation