Tau seed amplification assay reveals relationship between seeding and pathological forms of tau in Alzheimer's disease brain.

Tau seed amplification assays (SAAs) directly measure the seeding activity of tau and would therefore be ideal biomarkers for clinical trials targeting seeding-competent tau in Alzheimer's disease (AD). However, the precise relationship between tau seeding measured by SAA and the levels of pathological forms of tau in the AD brain remains unknown. We developed a new tau SAA based on full-length 0N3R tau with sensitivity in the low fg/ml range and used it to characterize 103 brain samples from three independent cohorts. Tau seeding clearly discriminated between AD and control brain samples. Interestingly, seeding was absent in Progressive Supranuclear Palsy (PSP) putamen, suggesting that our tau SAA did not amplify 4R tau aggregates from PSP brain. The specificity of our tau SAA for AD brain was further supported by analysis of matched hippocampus and cerebellum samples. While seeding was detected in hippocampus from Braak stages I-II, no seeding was present in AD cerebellum that is devoid of tau inclusions. Analysis of 40 middle frontal gyrus samples encompassing all Braak stages showed that tau SAA seeding activity gradually increased with Braak stage. This relationship between seeding activity and the presence of tau inclusions in AD brain was further supported by robust correlations between tau SAA results and the levels of phosphorylated tau212/214, phosphorylated tau181, aggregated tau, and sarkosyl-insoluble tau. Strikingly, we detected tau seeding in the middle frontal gyrus already at Braak stage II-III, suggesting that tau SAA can detect tau pathology earlier than conventional immunohistochemical staining. In conclusion, our data suggest a quantitative relationship between tau seeding activity and pathological forms of tau in the human brain and provides an important basis for further development of tau SAA for accessible human samples.

Effect of Repeated Administration of ɣ-Valerolactone (GVL) and GHB in the Mouse: Neuroadaptive Changes of the GHB and GABAergic System.

BACKGROUND: Gamma-hydroxybutyric acid (GHB) at low dosages has anxiolytic effects and promotes REM sleep and low-wave deep sleep. In the U.S., the legal form of GHB is prescribed to adults suffering from narcolepsy-associated cataplexy; the sodium salt of GHB is reserved for alcohol-addiction treatment. GHB is also a molecule of abuse and recreational use, it is a controlled substance in several countries, so gamma-valerolactone (GVL) has frequently been used as a legal substitute for it. GHB's abuse profile is most likely attributable to its anxiolytic, hypnotic, and euphoric properties, as well as its widespread availability and inexpensive/low cost on the illicit market. METHODS: Our study is focused on evaluating the potential effects on the mouse brain after repeated/prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) through behavioral study and immunohistochemical analysis using the markers tetraspanin 17 (TSPAN17), aldehyde dehydrogenase 5 (ALDH5A1), Gamma-aminobutyric acid type A receptor (GABA-A), and Gamma-aminobutyric acid type B receptor (GABA-B). RESULTS: Our findings revealed that prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) can have effects on a component of the mouse brain, the intensity of which can be assessed using immunohistochemistry. The findings revealed that long-term GHB administration causes a significant plastic alteration of the GHB signaling system, with downregulation of the putative binding site (TSPAN17) and overexpression of ALDH5A1, especially in hippocampal neurons. Our findings further revealed that GABA-A and GABA-B receptors are downregulated in these brain locations, resulting in a greater decrease in GABA-B expression. CONCLUSIONS: The goal of this study, from the point of view of forensic pathology, is to provide a new methodological strategy for better understanding the properties of this controversial substance, which could help us better grasp the unknown mechanism underlying its abuse profile.

Fully co-factor-free ClearTau platform produces seeding-competent Tau fibrils for reconstructing pathological Tau aggregates.

Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients' brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method - ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.

Dual truncation of tau by caspase-2 accelerates its CHIP-mediated degradation.

Intraneuronal aggregates of the microtubule binding protein Tau are a hallmark of different neurodegenerative diseases including Alzheimer's disease (AD). In these aggregates, Tau is modified by posttranslational modifications such as phosphorylation as well as by proteolytic cleavage. Here we identify a novel Tau cleavage site at aspartate 65 (D65) that is specific for caspase-2. In addition, we show that the previously described cleavage site at D421 is also efficiently processed by caspase-2, and both sites are cleaved in human brain samples. Caspase-2-generated Tau fragments show increased aggregation potential in vitro, but do not accumulate in vivo after AAV-mediated overexpression in mouse hippocampus. Interestingly, we observe that steady-state protein levels of caspase-2 generated Tau fragments are low in our in vivo model despite strong RNA expression, suggesting efficient clearance. Consistent with this hypothesis, we find that caspase-2 cleavage significantly improves the recognition of Tau by the ubiquitin E3 ligase CHIP, leading to increased ubiquitination and faster degradation of Tau fragments. Taken together our data thus suggest that CHIP-induced ubiquitination is of particular importance for the clearance of caspase-2 generated Tau fragments in vitro and in vivo.

Tau in the brain interstitial fluid is fragmented and seeding-competent.

In Alzheimer disease, Tau pathology is thought to propagate from cell to cell throughout interconnected brain areas. However, the forms of Tau released into the brain interstitial fluid (ISF) in vivo during the development of Tauopathy and their pathological relevance remain unclear. Combining in vivo microdialysis and biochemical analysis, we find that in Tau transgenic mice, human Tau (hTau) present in brain ISF is truncated and comprises at least 10 distinct fragments spanning the entire Tau protein. The fragmentation pattern is similar across different Tau transgenic models, pathological stages and brain areas. ISF hTau concentration decreases during Tauopathy progression, while its phosphorylation increases. ISF from mice with established Tauopathy induces Tau aggregation in HEK293-Tau biosensor cells. Notably, immunodepletion of ISF phosphorylated Tau, but not Tau fragments, significantly reduces its ability to seed Tau aggregation and only a fraction of Tau, separated by ultracentrifugation, is seeding-competent. These results indicate that ISF seeding competence is driven by a small subset of Tau, which potentially contribute to the propagation of Tau pathology.

SETD7-mediated monomethylation is enriched on soluble Tau in Alzheimer's disease.

BACKGROUND: Human tauopathies including Alzheimer's disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance. METHODS: We used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons. RESULTS: Our study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130. CONCLUSIONS: Our findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon.

Cell-cell coupling and DNA methylation abnormal phenotypes in the after-hours mice.

BACKGROUND: DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. METHODS: In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. RESULTS: We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. CONCLUSIONS: The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

Quantification of 1,3-ß-d-glucan by Wako ß-glucan assay for rapid exclusion of invasive fungal infections in critical patients: A diagnostic test accuracy study.

OBJECTIVES: Rapid and reliable exclusion of invasive fungal infections (IFI) by markers able to avoid unnecessary empirical antifungal treatment is still a critical unmet clinical need. We investigated the diagnostic performance of a newly available ß-d-Glucan (BDG) quantification assay, focusing on the optimisation of the BDG cut-off values for IFI exclusion. METHODS: BDG results by Wako ß-glucan assay (lower limit of detection [LLOD] = 2.16 pg/mL, positivity ≥ 11 pg/mL) on two consecutive serum samples were retrospectively analysed in 170 patients, admitted to haematological wards (N = 42), intensive care units (ICUs; N = 80), or other wards (N = 48), exhibiting clinical signs and/or symptoms suspected for IFI. Only patients with proven IFI (EORTC/MSG criteria) were considered as true positives in the assessment of BDG sensitivity, specificity and predictive values. RESULTS: Patients were diagnosed with no IFI (69.4%), proven IFI (25.3%) or probable IFI (5.3%). Two consecutive BDG values < LLOD performed within a median of 1 (interquartile range: 1-3) day were able to exclude a proven IFI with 100% sensitivity and negative predictive value (primary study goal). Test's specificity improved by using two distinct positivity and negativity cut-offs (7.7 pg/mL and LLOD, respectively), but remained suboptimal in ICU patients (50%), as compared to haematological or other patients (93% and 90%, respectively). CONCLUSIONS: The classification of Wako's results as negative when < LLOD, and positive when > 7.7 pg/mL, could be a promising diagnostic approach to confidently rule out an IFI in both ICU and non-ICU patients. The poor specificity in the ICU setting remains a concern, due to the difficulty to interpret positive results in this fragile population.

N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain.

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

Intensive multidisciplinary management in critical care patients affected by severe necrotizing soft tissue infections: a cooperative method to improve the efficacy of treatment.

To illustrate the effectiveness of our intensive multidisciplinary management (IMM) in the treatment of severely ill patients with necrotizing soft tissue infections (NSTIs). A retrospective observational study was conducted in a general ICU. Thirty-two consecutive patients undergoing IMM were carefully compared with 30 consecutive patients receiving a standard management (SM). IMM combined intensive care management, early surgical debridement followed by daily inspection of surgical wounds, close microbiological surveillance, and targeted high-dose antibiotics. IMM was associated with the better decrease of daily SOFA score (p = 0.04). Also, IMM caused + 12% increase in the overall number of surgical procedures (p = 0.022) and a higher number of tissue biopsies/per day (median 0.63 versus 0.32; p = 0.025), leading to a more targeted antimicrobial changes (89.6% vs 51.6%; p < 0.00001). High-dose daptomycin (75% vs 36.7%; p = 0.002) and extended/continuous infusion of beta-lactams (75% vs 43.3%; p = 0.011) were more frequently utilized. A specific efficiency score correlated with the decrease of SOFA score (efficacy) in IMM patients only (p = 0.027). Finally, IMM was associated with a significant lower ICU mortality rate (15.6% vs 40%; p = 0.032). IMM was more effective than SM as it allowed the earlier control of infection and the faster reduction of multiple organ-dysfunction.

Mice overexpressing lamin B1 in oligodendrocytes recapitulate the age-dependent motor signs, but not the early autonomic cardiovascular dysfunction of autosomal-dominant leukodystrophy (ADLD).

Autosomal dominant leukodystrophy (ADLD) is a rare adult-onset demyelinating disease caused by overexpression of lamin B1, a nuclear lamina filament. Early autonomic dysfunction involving the cardiovascular system before progressive somatic motor dysfunction is a striking feature of most cases of ADLD. In the Plp-FLAG-LMNB1 transgenic mouse model, lamin B1 overexpression in oligodendrocytes elicits somatic motor dysfunction and neuropathology akin to ADLD. Here, we investigate whether Plp-FLAG-LMNB1 mice also develop autonomic cardiovascular dysfunction before or after somatic motor dysfunction. We find that Plp-FLAG-LMNB1 mice have preserved cardiovascular responses to changes in wake-sleep state and ambient temperature and normal indexes of autonomic modulation at 37-42weeks of age despite a progressive somatic motor dysfunction, which includes impairments of walking ability (the ability to walk on a narrow path was impaired in 80% of mice at 34-38weeks of age) and subtle breathing derangements. Only late in the development of the disease phenotype did Plp-FLAG-LMNB1 mice develop a structural deficit of sympathetic noradrenergic fibers, with a 38% decrease in fiber profiles in the kidneys at 44-47weeks of age. We demonstrate that while the Plp-FLAG-LMNB1 mouse model recapitulates the age-dependent motor dysfunction of ADLD, it does not show signs of early autonomic cardiovascular dysfunction, raising the possibility that oligodendrocyte dysfunction may not be sufficient to cause the full spectrum of clinical features present in ADLD.

Lamin B1 levels modulate differentiation into neurons during embryonic corticogenesis.

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development. Ablation of endogenous Lamin B1 (Lmnb1) in the mouse strongly impairs embryonic brain development and corticogenesis. However, the mechanisms underlying these neurodevelopmental effects are unknown. Here, we report that Lamin B1 levels modulate the differentiation of murine neural stem cells (NSCs) into neurons and astroglial-like cells. In vitro, endogenous Lmnb1 depletion favors NSC differentiation into glial fibrillar acidic protein (GFAP)-immunoreactive cells over neurons, while overexpression of human Lamin B1 (LMNB1) increases the proportion of neurons. In Lmnb1-null embryos, neurogenesis is reduced, while in vivo Lmnb1 silencing in mouse embryonic brain by in utero electroporation of a specific Lmnb1 sh-RNA results in aberrant cortical positioning of neurons and increased expression of the astrocytic marker GFAP in the cortex of 7-day old pups. Together, these results indicate that finely tuned levels of Lamin B1 are required for NSC differentiation into neurons, proper expression of the astrocytic marker GFAP and corticogenesis.

Septic shock due to NSTI caused by Actinomyces Turicensis: the role of clinical pharmacology. Case report and review of the literature.

Critical septic patients affected by necrotizing soft tissue infections (NSTI) require an early, aggressive and multidisciplinary treatment. Pharmacokinetic alterations in antibiotic therapy are peculiar in these infections. Clinical pharmacology represents a first step in this setting. We report a case of septic shock due to NSTI in which clinical pharmacology is taken into account.

Advanced imaging of tau pathology in Alzheimer Disease: New perspectives from super resolution microscopy and label-free nanoscopy.

Alzheimer's disease (AD) is the main cause of dementia in the elderly population. Over 30 million people worldwide are living with dementia and AD prevalence is projected to increase dramatically in the next two decades. In terms of neuropathology, AD is characterized by two major cerebral hallmarks: extracellular ß-amyloid (Aß) plaques and intracellular Tau inclusions, which start accumulating in the brain 15-20 years before the onset of symptoms. Within this context, the scientific community worldwide is undertaking a wide research effort to detect AD pathology at its earliest, before symptoms appear. Neuroimaging of Aß by positron emission tomography (PET) is clinically available and is a promising modality for early detection of Aß pathology and AD diagnosis. Substantive efforts are ongoing to develop advanced imaging techniques for early detection of Tau pathology. Here, we will briefly describe the key features of Tau pathology and its heterogeneity across various neurodegenerative diseases bearing cerebral Tau inclusions (i.e., tauopathies). We will outline the current status of research on Tau-specific PET tracers and their clinical development. Finally, we will discuss the potential application of novel super-resolution and label-free techniques for investigating Tau pathology at the experimental level and their potential application for AD diagnosis. Microsc. Res. Tech. 79:677-683, 2016. © 2016 Wiley Periodicals, Inc.

Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy.

BACKGROUND: Alzheimer disease (AD) and other tauopathies develop cerebral intracellular inclusions of hyperphosphorylated tau. Epidemiological and experimental evidence suggests a clear link between type 2 diabetes mellitus and AD. In AD animal models, tau pathology is exacerbated by metabolic comorbidities, such as insulin resistance and diabetes. Within this context, anitidiabetic drugs, including the widely-prescribed insulin-sensitizing drug metformin, are currently being investigated for AD therapy. However, their efficacy for tauopathy in vivo has not been tested. RESULTS: Here, we report that in the P301S mutant human tau (P301S) transgenic mouse model of tauopathy, chronic administration of metformin exerts paradoxical effects on tau pathology. Despite reducing tau phosphorylation in the cortex and hippocampus via AMPK/mTOR and PP2A, metformin increases insoluble tau species (including tau oligomers) and the number of inclusions with ß-sheet aggregates in the brain of P301S mice. In addition, metformin exacerbates hindlimb atrophy, increases P301S hyperactive behavior, induces tau cleavage by caspase 3 and disrupts synaptic structures. CONCLUSIONS: These findings indicate that metformin pro-aggregation effects mitigate the potential benefits arising from its dephosphorylating action, possibly leading to an overall increase of the risk of tauopathy in elderly diabetic patients.

Tau-Driven Neuronal and Neurotrophic Dysfunction in a Mouse Model of Early Tauopathy.

Tauopathies are neurodegenerative diseases characterized by intraneuronal inclusions of hyperphosphorylated tau protein and abnormal expression of brain-derived neurotrophic factor (BDNF), a key modulator of neuronal survival and function. The severity of both these pathological hallmarks correlate with the degree of cognitive impairment in patients. However, how tau pathology specifically modifies BDNF signaling and affects neuronal function during early prodromal stages of tauopathy remains unclear. Here, we report that the mild tauopathy developing in retinal ganglion cells (RGCs) of the P301S tau transgenic (P301S) mouse induces functional retinal changes by disrupting BDNF signaling via the TrkB receptor. In adult P301S mice, the physiological visual response of RGCs to pattern light stimuli and retinal acuity decline significantly. As a consequence, the activity-dependent secretion of BDNF in the vitreous is impaired in P301S mice. Further, in P301S retinas, TrkB receptors are selectively upregulated, but uncoupled from downstream extracellular signal-regulated kinase (ERK) 1/2 signaling. We also show that the impairment of TrkB signaling is triggered by tau pathology and mediates the tau-induced dysfunction of visual response. Overall our results identify a neurotrophin-mediated mechanism by which tau induces neuronal dysfunction during prodromal stages of tauopathy and define tau-driven pathophysiological changes of potential value to support early diagnosis and informed therapeutic decisions. SIGNIFICANCE STATEMENT: This work highlights the potential molecular mechanisms by which initial tauopathy induces neuronal dysfunction. Combining clinically used electrophysiological techniques (i.e., electroretinography) and molecular analyses, this work shows that in a relevant model of early tauopathy, the retina of the P301S mutant human tau transgenic mouse, mild tau pathology results in functional changes of neuronal activity, likely due to selective impairment of brain-derived neurotrophic factor signaling via its receptor, TrkB. These findings may have important translational implications for early diagnosis in a subset of Alzheimer's disease patients with early visual symptoms and emphasize the need to clarify the pathophysiological changes associated with distinct tauopathy stages to support informed therapeutic decisions and guide drug discovery.

Lamin B1 protein is required for dendrite development in primary mouse cortical neurons.

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 (LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution.

NO-donor thiacarbocyanines as multifunctional agents for Alzheimer's disease.

Some symmetrical and unsymmetrical thiacarbocyanines bearing NO-donor nitrooxy and furoxan moieties were synthesized and studied as candidate anti-Alzheimer's drugs. All products activated soluble guanylate cyclase (sGC) in a dose-dependent manner, depending on the presence in their structures of NO-donor groups. None displayed toxicity when tested at concentrations below 10 µM on human brain microvascular endothelial cells (hCMEC/D3). Some products were capable of inhibiting amyloid ß-protein (Aß) aggregation, with a potency in the low µM concentration range, and of inhibiting aggregation of human recombinant tau protein in amyloid fibrils when incubated with the protein at 1 µM concentration. Nitrooxy derivative 21 and furoxan derivative 22 were selected to investigate synaptic plasticity. Both products, tested at 2 µM concentration, counteracted the inhibition of long-term potentiation (LTP) induced by Aß42 in hippocampal brain slices.

Messenger RNA processing is altered in autosomal dominant leukodystrophy.

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.