Phenothiazines to Treat Alzheimer’s Disease

Current treatments of Alzheimer’s Disease (AD) are largely ineffective and do not address underlying pathophysiological processes. The model organism C. elegans has been successfully used to discover compounds to treat human diseases, some now in clinical trials. To develop novel drugs and explore pathways to treat AD, we took on a forward pharmacological approach with a C. elegans model for AD, completed with studies to expand results to lifespan as well as healthspan. We screened 2560 drugs from the Microsource Spectrum library for their ability to delay proteotoxicity (indicated by paralysis) in an Abeta transgenic C. elegans muscle model of AD (CL2006) in liquid medium. Among the most protective drugs were phenothiazines, which are orally active and cross the blood-brain barrier, desirable properties of drugs to treat AD. 80 phenothiazines congeners were further assessed; 60% were protective in CL2006 worms. 9/20 tested phenothiazines increased lifespan in N2 worms and 2/3 phenothiazines tested promoted significantly higher pharyngeal pumping rates compared with control till day 10 of adulthood in N2 worms. 2 of the drugs were protective in the C. elegans neuronal model of AD. This phenotypic screening approach led to the discovery of potential drugs to treat AD. These phenothiazines protect against Abeta toxicity, and assessment of efficacy to protect against other forms of proteotoxicity are ongoing. These studies suggest the utility of C. elegans to discover drugs to treat human diseases. Future studies will assess molecular mechanisms mediating the protective effects of these compounds.

Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays

Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe techniques to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 h improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of our techniques by optimising it further for neurite outgrowth analysis. The presented methods achieve homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.

Multiple and Complex Codimension-2 Bifurcations underlying Post-inhibitory Rebound Spike and Excitability Transition

Neuron exhibits nonlinear dynamics such as excitability transition and post-inhibitory rebound (PIR) spike related to bifurcations, which are associated with information processing, locomotor modulation, or brain disease. PIR spike is evoked by inhibitory stimulation instead of excitatory stimulation, which presents a challenge to the threshold concept. In the present paper, 7 codimension-2 or degenerate bifurcations related to 10 codimension-1 bifurcations are acquired in a neuronal model, which presents the bifurcations underlying the excitability transition and PIR spike. Type I excitability corresponds to saddle-node bifurcation on an invariant cycle (SNIC) bifurcation, and type II excitability to saddle-node (SN) bifurcation or sub-critical Hopf (SubH) bifurcation or sup-critical Hopf (SupH) bifurcation. The excitability transition from type I to II corresponds to the codimension-2 bifurcation, Saddle-Node Homoclinic orbit (SNHO) bifurcation, via which SNIC bifurcation terminates and meanwhile big homoclinic orbit (BHom) bifurcation and SN bifurcation emerge. A degenerate bifurcation via which BHom bifurcation terminates and fold limit cycle (LPC) bifurcation emerges is responsible for spiking transition from type I to II, and the roles of other codimension-2 bifurcations (Cusp, Bogdanov-Takens, and Bautin) are discussed. In addition, different from the widely accepted viewpoint that PIR spike is mainly evoked near Hopf bifurcation rather than SNIC bifurcation, PIR spike is identified to be induced near SNIC or BHom or LPC bifurcations, and threshold curves resemble that of Hopf bifurcation. The complex bifurcations present comprehensive and deep understandings of excitability transition and PIR spike, which are helpful for the modulation to neural firing activities and physiological functions.

Diverse Dynamic Behaviors and Firing Activities of the Modified Fractional-Order Hindmarsh–Rose Neuronal Model Induced by Fractional-Order

It is important to investigate the firing activities of neurons, and previous experimental works have shown that fractional-order neuronal models depict the firing rate of neurons more verifiably. In this study, a modified fractional-order Hindmarsh–Rose neuronal model is proposed, and the dynamics and firing activities are investigated. Some novel phenomenon can be found. First, by analyzing numerically and theoretically, the Hopf bifurcation is found to occur when the external direct current stimulus is chosen appropriately. The effects of fractional-order on the bifurcation are also studied. Second, when injecting a direct current stimulus, compared with the integer-order model, the system has more varying dynamic behaviors and firing pattern transitions. Under different external current stimulus, periodic firing patterns and chaotic firing patterns occur when fractional-order changes, but the regions of chaotic firing patterns are different. In other words, the transition mode of periodic firing and chaotic firing induced by fractional-order is different under different external current stimulus. The two-dimensional colored diagram of firing patterns is also investigated. Finally, when injecting periodic current stimulus, regular/irregular bursting, multiple spiking, regular\irregular square wave bursting, and mixed firing mods are found by setting the appropriate fractional-order, amplitude, and frequency of the external current stimulus. Some firing patterns cannot be found in integer-order models. When the amplitude is chosen at appropriate values, the region of frequency when the system displays the mixed firing modes decreases with increasing fractional-order.

Smart Regions in the Italian Context. From a Theoretical to an Empirical Framework

The present study aims to examine the role the social and digital infrastructures might have during the building process of the Smart Regions in the Italian context. Within this framework, it is possible to identify some essential research questions, such as why the same regions are growing faster than the other and which type of effects could be generated from the different connectivity between the regions. Since the Smart Region concept is still composed of technical reports, pilot projects and experiences from a limited number of cities on the international stage, this work it is tried to use a new approach, applying either a neuronal model, the Self-Organizing Maps, and the multivariate regression approach, to extrapolate the existence of possible future conditions for the rising of Smart Regions in Italy, studying the evolution of the used database during the period 2005 – 2016. From the analysis what emerged is that the only bridging social capital dimension, empirically speaking, feed the regional innovation growth because the structure of social relationship facilitates interactions across social, political and economic agents; but there are institutional deficits, most pronounced in Italy and other European countries.

A human iPSC-derived inducible neuronal model of Niemann-Pick disease, type C1

Background Niemann-Pick disease, type C (NPC) is a childhood-onset, lethal, neurodegenerative disorder caused by autosomal recessive mutations in the genes NPC1 or NPC2 and characterized by impaired cholesterol homeostasis, a lipid essential for cellular function. Cellular cholesterol levels are tightly regulated, and mutations in either NPC1 or NPC2 lead to deficient transport and accumulation of unesterified cholesterol in the late endosome/lysosome compartment, and progressive neurodegeneration in affected individuals. Previous cell-based studies to understand the NPC cellular pathophysiology and screen for therapeutic agents have mainly used patient fibroblasts. However, these do not allow modeling the neurodegenerative aspect of NPC disease, highlighting the need for an in vitro system that permits understanding the cellular mechanisms underlying neuronal loss and identifying appropriate therapies. This study reports the development of a novel human iPSC-derived, inducible neuronal model of Niemann-Pick disease, type C1 (NPC1). Results We generated a null i3Neuron (inducible × integrated × isogenic) (NPC1−/− i3Neuron) iPSC-derived neuron model of NPC1. The NPC1−/− and the corresponding isogenic NPC1+/+ i3Neuron cell lines were used to efficiently generate homogenous, synchronized neurons that can be used in high-throughput screens. NPC1−/− i3Neurons recapitulate cardinal cellular NPC1 pathological features including perinuclear endolysosomal storage of unesterified cholesterol, accumulation of GM2 and GM3 gangliosides, mitochondrial dysfunction, and impaired axonal lysosomal transport. Cholesterol storage, mitochondrial dysfunction, and axonal trafficking defects can be ameliorated by treatment with 2-hydroxypropyl-β-cyclodextrin, a drug that has shown efficacy in NPC1 preclinical models and in a phase 1/2a trial. Conclusion Our data demonstrate the utility of this new cell line in high-throughput drug/chemical screens to identify potential therapeutic agents. The NPC1−/− i3Neuron line will also be a valuable tool for the NPC1 research community to explore the pathological mechanisms contributing to neuronal degeneration. Graphical abstract

3F(eature)s model: modularity, heterogeneity and three-dimensionality to design in vitro neuronal model

In this work, we present a novel experimental platform to build in vitro interconnected (i.e., modular) heterogeneous (e.g., cortical-hippocampal) and three-dimensional (3D) neuronal cultures plated on Micro-Electrode Arrays (MEAs) to extracellularly record the electrophysiological activity continuously.

Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement

Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement—a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.