Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here ...we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.
Colasante et al. exploit an activatory CRISPR-targeting Scn1a gene promoter as a therapeutic strategy to rescue Scn1a haploinsufficiency in a mouse model of Dravet syndrome and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel.
Lake Maggiore has reached a stable oligotrophic status after a recovery process from mesotrophy that began in the early 1980s. However, the most recent phytoplankton and water chemistry data seem to ...indicate that various changes are taking place, including a slight increase in TP values, and almost regular blooms have occurred since 2005. Meteoclimatic data collected over the last few decades in the Lake Maggiore watershed highlight an increase in the frequency of extreme rainfall events. Here, for the period 2000–2013, we analyse the relationships between the phytoplankton dynamics and selected extreme precipitation events, in order to evaluate the possible role of rainfall in affecting the nutrient availability and phytoplankton dynamics. Among the algal groups, cyanobacteria showed the strongest relationship with the precipitation pattern, particularly on a short-term temporal scale. Our results support the hypothesis that rainfall may lead to a short-term increase in nutrients that stimulate the growth of phytoplankton and the development of blooms in summer, when epilimnetic waters are usually nutrient depleted. The almost regular cyanobacterial blooms recorded in Lake Maggiore since 2005 can be interpreted as a response to changing precipitation patterns and therefore as a sign of climate-induced eutrophication.
Dysfunctions in mitochondrial dynamics and metabolism are common pathological processes associated with Parkinson’s disease (PD). It was recently shown that an inherited form of PD and dementia is ...caused by mutations in the OPA1 gene, which encodes for a key player in mitochondrial fusion and structure. iPSC-derived neural cells from these patients exhibited severe mitochondrial fragmentation, respiration impairment, ATP deficits, and heightened oxidative stress. Reconstitution of normal levels of OPA1 in PD-derived neural cells normalized mitochondria morphology and function. OPA1-mutated neuronal cultures showed reduced survival in vitro. Intriguingly, selective inhibition of necroptosis effectively rescued this survival deficit. Additionally, dampening necroptosis in MPTP-treated mice protected from DA neuronal cell loss. This human iPSC-based model captures both early pathological events in OPA1 mutant neural cells and the beneficial effects of blocking necroptosis, highlighting this cell death process as a potential therapeutic target for PD.
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•OPA1 mutant iPSC-derived NPCs contain dysfunctional mitochondria•OPA1 mutant iPSC-derived NPCs present high levels of oxidative stress•Nec-1s can improve survival of OPA1 mutant human neurons in vitro•Nec-1s counteracts the dopaminergic cell loss in MPTP-treated neurons
Iannielli et al. generate iPSCs from Parkinson’s disease patients with OPA1 mutations and find that derived NPCs have mitochondria with impaired morphology and bioenergetics. Nec-1s, a pharmacological inhibitor of necroptosis, promotes the survival of human OPA1 mutant neurons and attenuates dopaminergic neuronal loss in MPTP-treated mice.
An original multivariate multi-scale methodology for assessing the complexity of physiological signals is proposed. The technique is able to incorporate the simultaneous analysis of multi-channel ...data as a unique block within a multi-scale framework. The basic complexity measure is done by using Permutation Entropy, a methodology for time series processing based on ordinal analysis. Permutation Entropy is conceptually simple, structurally robust to noise and artifacts, computationally very fast, which is relevant for designing portable diagnostics. Since time series derived from biological systems show structures on multiple spatial-temporal scales, the proposed technique can be useful for other types of biomedical signal analysis. In this work, the possibility of distinguish among the brain states related to Alzheimer’s disease patients and Mild Cognitive Impaired subjects from normal healthy elderly is checked on a real, although quite limited, experimental database.
Climate warming can restructure lake food webs if trophic levels differ in their thermal responses, but evidence for these changes and their underlying mechanisms remain scarce in nature. Here we ...document how warming lake temperatures by up to 2°C, rather than changes in trophic state or fishing effort, have restructured the pelagic food web of a large European lake (Lake Maggiore, Italy). Our approach exploited abundance and biomass data collected weekly to yearly across five trophic levels from 1981 to 2008. Temperature generally had stronger effects on taxa than changes in fish predation or trophic state mediated through primary productivity. Consequently, we found that, as the lake warmed, the food web shifted in numerical abundance towards predators occupying middle trophic positions. Of these taxa, the spiny water flea (Bythotrephes longimanus) most prospered. Bythotrephes strongly limited abundances of the keystone grazer Daphnia, strengthening top‐down structuring of the food web. Warmer temperatures partly restructured the food web by advancing peak Bythotrephes densities by approximately 60 days and extending periods of positive population growth by three times. Nonetheless, our results suggested that advances in the timing and size of peak Bythotrephes densities could not outpace changes in the timing and size of peak densities in their Daphnia prey. Our results provide rare evidence from nature as to how long‐term warming can favour higher trophic levels, with the potential to strengthen top‐down control of food webs.
We provide rare evidence from nature of how long‐term climate warming restructures food webs and their phenology. By analysing exceptionally long time series in an iconic European lake, we found predators most benefited from warming, partly by peaking in density earlier and experiencing a longer growing season. The effects of warming exceeded those of other global changes, namely in nutrient status and overfishing, and ultimately strengthened top‐down control over the lake food web.
Increases in atmospheric temperature and nutrients from land are thought to be promoting the expansion of harmful cyanobacteria in lakes worldwide, yet to date there has been no quantitative ...synthesis of long‐term trends. To test whether cyanobacteria have increased in abundance over the past ~ 200 years and evaluate the relative influence of potential causal mechanisms, we synthesised 108 highly resolved sedimentary time series and 18 decadal‐scale monitoring records from north temperate‐subarctic lakes. We demonstrate that: (1) cyanobacteria have increased significantly since c. 1800 ce, (2) they have increased disproportionately relative to other phytoplankton, and (3) cyanobacteria increased more rapidly post c. 1945 ce. Variation among lakes in the rates of increase was explained best by nutrient concentration (phosphorus and nitrogen), and temperature was of secondary importance. Although cyanobacterial biomass has declined in some managed lakes with reduced nutrient influx, the larger spatio‐temporal scale of sedimentary records show continued increases in cyanobacteria throughout the north temperate‐subarctic regions.
Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles ...since
is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone
(i
) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral
transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic
mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female
mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male
mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of i
provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.
The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by ...intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.
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Extensive gene delivery in the CNS is attainable through a single systemic injection of AAV-PHP.B. The authors exploited this system to simultaneously target different brain areas and modulate their functions but also to unveil its therapeutic potential. Global transduction of a therapeutic gene reversed pathological symptoms in a model of synucleinopathy.
•A 1D coupled ecological-hydrodynamic model is applied to a very deep large lake.•Calibration and validation are performed on an overall 16.75-year period.•Predictions of deep-water chemistry and ...phytoplankton succession are evaluated.•Comparable to superior performances to those of previous studies are obtained.•Impossibility to reproduce shifts in plankton species hinders long-term prognoses.
One-dimensional coupled ecological-hydrodynamic numerical models of lakes require extensive calibration of their chemical and biological parameters. Application of these models to future projections relies on the time invariance of the calibrated model parameters and of the adopted schematisation. This is mere speculation for real ecosystems, so that it is relevant to explore the limits of coupled models over extended periods. To date, almost all applications in literature have been calibrated over a couple of years at most, with comparable validation periods, if present. Furthermore, past studies mostly concerned shallow to moderately deep small lakes, so that reproducing the hypolimnetic chemical evolution of very deep large lakes has generally been overlooked. Last, most works did not compare with observations or even model the succession of phytoplankton species, but only dealt with total Chlorophyll-a. Here, the GLM-AED2 (General Lake Model – Aquatic EcoDynamics) coupled model was calibrated and validated for an overall 16.75-year period for the 370-m deep and 213-km2 wide Lake Maggiore (Northern Italy/Southern Switzerland), focusing on the reproduction of both deep-water chemistry and phytoplankton biomass and succession. Despite the modelling simplifications needed for this complex basin, the resulting performances are comparable to those in literature for shallower and smaller lakes over shorter periods. Still, extreme care must be put when interpreting the results of coupled ecological-hydrodynamic models for long-term projections, especially regarding the evolution of phytoplankton.
Lake Orta (Northern Italy) became one of the world’s largest acidic lakes, following industrial pollution, beginning in the late 1920s. Prior to pollution, Lake Orta supported a rich and diversified ...phytoplankton community dominated by diatoms, cyanobacteria and dinoflagellates. Their taxonomic composition was comparable to that of the nearby Lake Maggiore, which provides a useful reference comparison. After pollution, Lake Orta was so acidic and contaminated with trace metals that only a few tolerant phytoplankton species persisted, supplemented by sudden and short living outbursts of occasional colonists. The lake was limed in 1989-1990. This has permitted the gradual recovery of its chemistry and biology, and many phytoplankton species that inhabit Lake Maggiore are now re-appearing in Lake Orta. I tested the two hypotheses that Lakes Orta and Maggiore would now have a similar phytoplankton taxonomic assemblages, and similar diversity of functional groups given their similar morphometry, physical features and trophic states. The two hypotheses were tested by comparing the phytoplankton assemblages of lakes Maggiore and Orta for the first 10 years after liming, i.e. 1990 to 2001. Phytoplankton was classified according the Reynolds' Morpho Functional Groups and five diversity indices were calculated (S, number of units; H, Shannon-Wiener; E, evenness; D, dominance; J, equitability). SHE analysis (an analysis of diversity changes based on the relationship among species richness (S), H Index (H) and evenness (E)) was also carried out, in order to compare the long term trend of both functional groups and taxa biodiversity. Both taxonomic and the functional composition differed in the two lakes, likely because chemical quality have played a role in taxaselection. Moreover, it was quite clear that, during the first post-liming decade, Lake Orta’s phytoplankton was characterized by low diversity and evenness and by marked year-to-year fluctuations. However, SHE analysis showed that the colonization rate was higher in Lake Orta than in Lake Maggiore, and that the environmental modifications caused by the liming were opening new ecological niches, allowing some colonists to thrive in the changing, albeit still unusual chemical environment of the lake.
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