Huntington's Disease Patient-Derived Astrocytes Display Electrophysiological Impairments and Reduced Neuronal Support.

In Huntington's disease (HD), while the ubiquitously expressed mutant Huntingtin (mtHTT) protein primarily compromises striatal and cortical neurons, glia also undergo disease-contributing alterations. Existing HD models using human induced pluripotent stem cells (iPSCs) have not extensively characterized the role of mtHTT in patient-derived astrocytes. Here physiologically mature astrocytes are generated from HD patient iPSCs. These human astrocytes exhibit hallmark HD phenotypes that occur in mouse models, including impaired inward rectifying K+ currents, lengthened spontaneous Ca2+ waves and reduced cell membrane capacitance. HD astrocytes in co-culture provided reduced support for the maturation of iPSC-derived neurons. In addition, neurons exposed to chronic glutamate stimulation are not protected by HD astrocytes. This iPSC-based HD model demonstrates the critical effects of mtHTT on human astrocytes, which not only broadens the understanding of disease susceptibility beyond cortical and striatal neurons but also increases potential drug targets.

Patients with ALS show highly correlated progression rates in left and right limb muscles.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) progresses at different rates between patients, making clinical trial design difficult and dependent on large cohorts of patients. Currently, there are few data showing whether the left and right limbs progress at the same or different rates. This study addresses rates of decline in specific muscle groups of patients with ALS and assesses whether there is a relationship between left and right muscles in the same patient, regardless of overall progression. METHODS: A large cohort of patients was used to assess decline in muscle strength in right and left limbs over time using 2 different methods: The Tufts Quantitative Neuromuscular Exam and Accurate Test of Limb Isometric Strength protocol. Then advanced linear regression statistical methods were applied to assess progression rates in each limb. RESULTS: This report shows that linearized progression models can predict general slopes of decline with good accuracy. Critically, the data demonstrate that while overall decline is variable, there is a high degree of correlation between left and right muscle decline in ALS. This implies that irrespective of which muscle starts declining soonest or latest, their rates of decline following onset are more consistent. CONCLUSIONS: First, this study demonstrates a high degree of power when using unilateral treatment approaches to detect a slowing in disease progression in smaller groups of patients, thus allowing for paired statistical tests. These findings will be useful in transplantation trials that use muscle decline to track disease progression in ALS. Second, these findings discuss methods, such as tactical selection of muscle groups, which can improve the power efficiency of all ALS clinical trials.

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA.

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.

Stimulation of GABA-induced Ca2+ influx enhances maturation of human induced pluripotent stem cell-derived neurons.

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca(2+) channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca(2+) imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca(2+) channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca(2+) or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca(2+) channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca(2+) channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.

Ectodomain shedding of the Notch ligand Jagged1 is mediated by ADAM17, but is not a lipid-raft-associated event.

Notch signalling is an evolutionarily conserved pathway involved in cell-fate specification. The initiating event in this pathway is the binding of a Notch receptor to a DSL (Delta/Serrate/Lag-2) ligand on neighbouring cells triggering the proteolytic cleavage of Notch within its extracellular juxtamembrane region; a process known as proteolytic 'shedding' and catalysed by members of the ADAM (a disintegrin and metalloproteinase) family of enzymes. Jagged1 is a Notch-binding DSL ligand which is also shed by an ADAM-like activity raising the possibility of bi-directional cell-cell Notch signalling. In the present study we have unequivocally identified the sheddase responsible for shedding Jagged1 as ADAM17, the activity of which has previously been shown to be localized within specialized microdomains of the cell membrane known as 'lipid rafts'. However, we have shown that replacing the transmembrane and cytosolic regions of Jagged1 with a GPI (glycosylphosphatidylinositol) anchor, thereby targeting the protein to lipid rafts, did not enhance its shedding. Furthermore, the Jagged1 holoprotein, its ADAM-cleaved C-terminal fragment and ADAM17 were not enriched in raft preparations devoid of contaminating non-raft proteins. We have also demonstrated that wild-type Jagged1 and a truncated polypeptide-anchored variant lacking the cytosolic domain were subject to similar constitutive and phorbol ester-regulated shedding. Collectively these data demonstrate that Jagged1 is shed by ADAM17 in a lipid-raft-independent manner, and that the cytosolic domain of the former protein is not a pre-requisite for either constitutive or regulated shedding.