Hyperexcitability in young iPSC-derived C9ORF72 mutant motor neurons is associated with increased intracellular calcium release

S.B. was supported by a Motor Neuron Disease Association studentship (WadeMartins/Oct13/867-792) and D.A.B was supported by the Monument Trust Discovery Award (J-1403) from Parkinson’s UK. The work was supported by the Medical Research Council Dementias Platform UK Stem Cell Network (Grant MR/M024962/1 to RW-M).A large hexanucleotide repeat expansion in the C9ORF72 gene is the most prevalent cause of amyotrophic lateral sclerosis (ALS). To better understand neuronal dysfunction during ALS progression, we studied motor neuron (MN) cultures derived from iPSC lines generated from C9ORF72 (C9) expansion carriers and unaffected controls. C9 and control MN cultures showed comparable mRNA levels for MN markers SMI-32, HB9 and ISL1 and similar MN yields (> 50% TUJ1/SMI-32 double-positive MNs). Using whole-cell patch clamp we showed that C9-MNs have normal membrane capacitance, resistance and resting potential. However, immature (day 40) C9-MNs exhibited a hyperexcitable phenotype concurrent with increased release of calcium (Ca2+) from internal stores, but with no changes to NaV and KV currents. Interestingly, this was a transient phenotype. By day 47, maturing C9-MNs demonstrated normal electrophysiological activity, displaying only subtle alterations on mitochondrial Ca2+ release. Together, these findings suggest the potential importance of a developmental component to C9ORF72-related ALS.Publisher PDFPeer reviewe

From synaptic dysfunction to neuroprotective strategies in genetic Parkinson’s disease: lessons from LRRK2

The pathogenesis of Parkinson’s disease (PD) is thought to rely on a complex interaction between the patient’s genetic background and a variety of largely unknown environmental factors. In this scenario, the investigation of the genetic bases underlying familial PD could unveil key molecular pathways to be targeted by new disease-modifying therapies, still currently unavailable. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are responsible for the majority of inherited familial PD cases and can also be found in sporadic PD, but the pathophysiological functions of LRRK2 have not yet been fully elucidated. Here, we will review the evidence obtained in transgenic LRRK2 experimental models, characterized by altered striatal synaptic transmission, mitochondrial dysfunction, and α-synuclein aggregation. Interestingly, the processes triggered by mutant LRRK2 might represent early pathological phenomena in the pathogenesis of PD, anticipating the typical neurodegenerative features characterizing the late phases of the disease. A comprehensive view of LRRK2 neuronal pathophysiology will support the possible clinical application of pharmacological compounds targeting this protein, with potential therapeutic implications for patients suffering from both familial and sporadic PD

Atypical progressive supranuclear palsy with corticospinal tract degeneration.

Progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), sporadic multisystem tauopathy, and some forms of frontotemporal dementia with Parkinsonism linked to chromosome 17 are characterized by neuronal and glial lesions accumulating tau protein containing 4 conserved repeats in microtubule-binding domain (4R tau). Corticospinal tract degeneration is not a common feature of 4R tauopathies. Our objective was to describe 12 cases with pathologic features similar to those of PSP but with prominent corticospinal tract degeneration. We reviewed the historical records and neuropathologic evaluation using standardized sampling, immunohistochemistry, semiquantitative analysis, image analysis, and electron microscopy. The mean age at onset and illness duration was 71 and 5.7 years, respectively. Eight cases were female. Eleven cases had clinical evidence of prominent upper motor neuron disease plus extrapyramidal features. There was focal parasagittal cortical atrophy involving motor cortex and degeneration of corticospinal tract with sparing of lower motor neurons like in primary lateral sclerosis. Prominent tau pathology was found in oligodendrocytes in motor cortex, subjacent white matter, and corticospinal tract characterized by globular cytoplasmic filamentous inclusions that were immunoreactive for 4R tau. The clinicopathologic features of these 12 cases expand the spectrum of 4R tauopathies

Calcium dysregulation combined with mitochondrial failure and electrophysiological maturity converge in Parkinson’s iPSC-dopamine neurons

Summary: Parkinson’s disease (PD) is characterized by a progressive deterioration of motor and cognitive functions. Although death of dopamine neurons is the hallmark pathology of PD, this is a late-stage disease process preceded by neuronal dysfunction. Here we describe early physiological perturbations in patient-derived induced pluripotent stem cell (iPSC)-dopamine neurons carrying the GBA-N370S mutation, a strong genetic risk factor for PD. GBA-N370S iPSC-dopamine neurons show an early and persistent calcium dysregulation notably at the mitochondria, followed by reduced mitochondrial membrane potential and oxygen consumption rate, indicating mitochondrial failure. With increased neuronal maturity, we observed decreased synaptic function in PD iPSC-dopamine neurons, consistent with the requirement for ATP and calcium to support the increase in electrophysiological activity over time. Our work demonstrates that calcium dyshomeostasis and mitochondrial failure impair the higher electrophysiological activity of mature neurons and may underlie the vulnerability of dopamine neurons in PD

Tau depletion in human neurons mitigates A?-driven toxicity

Alzheimer’s disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-β (Aβ) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aβ-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aβ were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effects by mitigating the impact of exogenous Aβ-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aβ-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt−/− mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types

Behavioral deficits and striatal DA signaling in LRRK2 p.G2019S transgenic rats: a multimodal investigation including PET neuroimaging

Background: A major risk-factor for developing Parkinson's disease (PD) is genetic variability in leucine-rich repeat kinase 2 (LRRK2), most notably the p.G2019S mutation. Examination of the effects of this mutation is necessary to determine the etiology of PD and to guide therapeutic development. Objective: Assess the behavioral consequences of LRRK2 p.G2019S overexpression in transgenic rats as they age and test the functional integrity of the nigro-striatal dopamine system. Conduct positron emission tomography (PET) neuroimaging to compare transgenic rats with previous data from human LRRK2 mutation carriers. Methods: Rats overexpressing human LRRK2 p.G2019S were generated by BAC transgenesis and compared to non-transgenic (NT) littermates. Motor skill tests were performed at 3, 6 and 12 months-of-age. PET, performed at 12 months, assessed the density of dopamine and vesicular monoamine transporters (DAT and VMAT2, respectively) and measured dopamine synthesis, storage and availability. Brain tissue was assayed for D2, DAT, dopamine and cAMP-regulated phosphoprotein (DARPP32) and tyrosine hydroxylase (TH) expression by Western blot, and TH by immunohistochemistry. Results: Transgenic rats had no abnormalities in measures of striatal dopamine function at 12 months. A behavioral phenotype was present, with LRRK2 p.G2019S rats performing significantly worse on the rotarod than non-transgenic littermates (26% reduction in average running duration at 6 months), but with normal performance in other motor tests. Conclusions: Neuroimaging using dopaminergic PET did not recapitulate prior studies in human LRRK2 mutation carriers. Consistently, LRRK2 p.G2019S rats do not develop overt neurodegeneration; however, they do exhibit behavioral abnormalities

Robotic multiwell planar patch-clamp for native and primary mammalian cells

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings