Altered calcium dynamics and glutamate receptor properties in iPSC-derived motor neurons from ALS patients with C9orf72, FUS, SOD1 or TDP43 mutations.

The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterized by a profound loss of motor neurons (MNs). Until now only riluzole minimally extends life expectancy in ALS, presumably by inhibiting glutamatergic neurotransmission and calcium overload of MNs. Therefore, the aim of this study was to investigate the glutamate receptor properties and key aspects of intracellular calcium dynamics in induced pluripotent stem cell (iPSC)-derived MNs from ALS patients with C9orf72 (n = 4 cell lines), fused in sarcoma (FUS) (n = 9), superoxide dismutase 1 (SOD1) (n = 3) or transactive response DNA-binding protein 43 (TDP43) (n = 3) mutations as well as healthy (n = 7 cell lines) and isogenic controls (n = 3). Using calcium imaging, we most frequently observed spontaneous transients in mutant C9orf72 MNs. Basal intracellular calcium levels and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced signal amplitudes were elevated in mutant TDP43 MNs. Besides, a majority of mutant TDP43 MNs responded to 3.5-dihydroxyphenylglycine as metabotropic glutamate receptor agonist. Quantitative real-time PCR demonstrated significantly increased expression levels of AMPA and kainate receptors in mutant FUS cells compared to healthy and isogenic controls. Furthermore, the expression of kainate receptors and voltage gated calcium channels in mutant C9orf72 MNs as well as metabotropic glutamate receptors in mutant SOD1 cells was markedly elevated compared to controls. Our data of iPSC-derived MNs from familial ALS patients revealed several mutation-specific alterations in glutamate receptor properties and calcium dynamics that could play a role in ALS pathogenesis and may lead to future translational strategies with individual stratification of neuroprotective ALS treatments.

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons.

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca2+ content and lower frequency of spontaneous Ca2+ signals in SGCE MSNs. Blocking of voltage-gated Ca2+ channels by verapamil was less efficient in suppressing KCl-induced Ca2+ peaks of SGCE MSNs. Ca2+ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca2+ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.

Generation of four iPSC lines from four patients with Leigh syndrome carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6.

We report the generation of four human iPSC lines (8993-A12, 8993-B12, 8993-C11, and 8993-D7) from fibroblasts of four patients affected by maternally inherited Leigh syndrome (MILS) carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6. We used Sendai viruses to deliver reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The established iPSC lines expressed pluripotency markers, exhibited a normal karyotype, were capable to form cells of the three germ layers in vitro, and retained the MT-ATP6 mutations at the same homoplasmic level of the parental fibroblasts.

Relationship Satisfaction in People with Parkinson's Disease and Their Caregivers: A Cross-Sectional Observational Study.

Parkinson's disease (PD) is a neurodegenerative disorder, which leads to reduced health-related quality of life (HR-QoL) and autonomy in advanced stages of the disease. Hence, people with PD (PwPD) are in need of help, which is often provided by informal caregivers, especially spouses. This might influence the relationship satisfaction in patients and their spousal caregivers. Additionally, previous studies have shown that a reduced relationship satisfaction may result in mental disorders and reduced physical health. The aim of this study is to identify factors influencing PwPD and their caregivers' relationship satisfaction in a cross-sectional observational study. Analyses revealed an overall satisfying relationship, measured by the Quality of Marriage Index, in PwPD (n = 84) and their caregivers (n = 79). Relationship satisfaction in PwPD mildly decreased with reduced HR-QoL and more severe depressive symptoms. Reduced relationship satisfaction in caregivers was significantly associated with decreased HR-QoL, higher caregiver burden, more severe depressive symptoms and increased neuropsychiatric symptoms in PwPD. Further studies are needed to investigate the influence of the identified factors over time and if relationship satisfaction has a reciprocal impact on caregiver burden, HR-QoL as well as mental and physical health.

Validation of the Parkinson's Disease Caregiver Burden Questionnaire in Progressive Supranuclear Palsy.

Progressive supranuclear palsy (PSP) is an atypical Parkinson syndrome with axial akinetic-rigid symptoms, early postural instability, and ocular motor impairments. Patients experience a rapid loss of autonomy and care dependency; thus, caregivers must assist in the activities of daily living early in the course of the disease. Caregiver burden is an extremely important factor in disease management. However, there are no specific questionnaires for assessment of caregiver burden in PSP. This study aims to validate the Parkinson's disease caregiver burden questionnaire (PDCB) as a specific measure of caregiver burden in PSP. PSP patients were assessed by the PSP rating scale, PSP quality-of-life questionnaire (PSP-QoL), Montreal cognitive assessment test (MoCA), and geriatric depression scale (GDS-15). Caregivers filled out the short form 36-health survey, GDS-15, PDCB, and the caregiver burden inventory (CBI). 22 patient caregiver pairs completed the study. PDCB showed a highly significant correlation with the CBI (r 0.911; p < 0.001). Internal reliability of the PDCB measured by Cronbach's alpha was favourable at 0.803. These data support the specificity of the PDCB in PSP caregivers. Future studies with larger sample sizes of PSP patients and caregivers and a multicentric longitudinal design should be performed to gain further insight of caregiver burden in PSP.

One Year Trajectory of Caregiver Burden in Parkinson's Disease and Analysis of Gender-Specific Aspects.

Parkinson's disease (PD) is a slowly progressive neurodegenerative movement disorder that leads to impairments in activities of daily living. In addition to reducing patients' quality of life, this disease also affects caregivers' well-being. Until recently, caregiver burden was mainly assessed by generic questionnaires, which do not take the characteristics of the chronic disease into consideration. In the case of PD, this issue has been addressed by the introduction of the "Parkinson's disease caregiver burden" questionnaire (PDCB). Data on longitudinal trajectories of caregiver burden are still missing in the literature. In this study, we assessed the one-year trajectory of caregiver burden by the PDCB as a disease-specific questionnaire. Further, gender-specific aspects of caregiver burden were analyzed by applying a caregiver task questionnaire. PDCB total score (n = 84 patients and caregivers) did not significantly change from baseline (30.4) to one year at follow-up (31.5). No significant difference was detected between female and male caregivers in global burden and-specific caregiver tasks. Our data showed only a mild increase of caregiver burden in the timeframe of one year. Gender-specific differences do not seem to impact-specific caregiver tasks in the presented study population.

Reduced Expression of GABA A Receptor Alpha2 Subunit Is Associated With Disinhibition of DYT-THAP1 Dystonia Patient-Derived Striatal Medium Spiny Neurons.

DYT-THAP1 dystonia (formerly DYT6) is an adolescent-onset dystonia characterized by involuntary muscle contractions usually involving the upper body. It is caused by mutations in the gene THAP1 encoding for the transcription factor Thanatos-associated protein (THAP) domain containing apoptosis-associated protein 1 and inherited in an autosomal-dominant manner with reduced penetrance. Alterations in the development of striatal neuronal projections and synaptic function are known from transgenic mice models. To investigate pathogenetic mechanisms, human induced pluripotent stem cell (iPSC)-derived medium spiny neurons (MSNs) from two patients and one family member with reduced penetrance carrying a mutation in the gene THAP1 (c.474delA and c.38G > A) were functionally characterized in comparison to healthy controls. Calcium imaging and quantitative PCR analysis revealed significantly lower Ca2+ amplitudes upon GABA applications and a marked downregulation of the gene encoding the GABA A receptor alpha2 subunit in THAP1 MSNs indicating a decreased GABAergic transmission. Whole-cell patch-clamp recordings showed a significantly lower frequency of miniature postsynaptic currents (mPSCs), whereas the frequency of spontaneous action potentials (APs) was elevated in THAP1 MSNs suggesting that decreased synaptic activity might have resulted in enhanced generation of APs. Our molecular and functional data indicate that a reduced expression of GABA A receptor alpha2 subunit could eventually lead to limited GABAergic synaptic transmission, neuronal disinhibition, and hyperexcitability of THAP1 MSNs. These data give pathophysiological insight and may contribute to the development of novel treatment strategies for DYT-THAP1 dystonia.

Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins.

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms.

Calcium, Sodium, and Transient Receptor Potential Channel Expression in Human Fetal Midbrain-Derived Neural Progenitor Cells.

Voltage-gated sodium and calcium channels as well as transient receptor potential (TRP) channels are expressed during the differentiation of human neural progenitor cells (hNPCs) and are likely to be involved in regulating neurogenesis. However, the molecular composition of these ion channels in proliferating and differentiating hNPCs is largely unknown. In this study, we investigated fetal mesencephalic hNPCs in respect to their sodium, calcium, and TRP channel subunit expression and function. Quantitative real-time polymerase chain reaction indicated a significant upregulation of voltage-gated sodium and calcium channel subunits in hNPCs after differentiation for 3 weeks in vitro. In contrast, the TRP channel expression did not increase significantly during hNPC maturation. Intracellular Ca2+ measurements showed the marked reduction of KCl-induced Ca2+ transients through inhibition of voltage-gated Ca2+ channels by verapamil and mibefradil in differentiated hNPCs. Application of TRP channel agonists induced intracellular Ca2+ peaks already in proliferating hNPCs without affecting their cell division. The coincubation of hNPCs with TRP channel agonists pregnenolone sulfate or RN1747 did not have any significant effect on their proliferation and differentiation. These data indicate that hNPCs derived from fetal midbrain tissue acquire essential voltage-gated sodium and calcium channel properties during neuronal maturation in vitro. An early role of TRP channels in neurogenesis which may be important for regenerative clinical applications or cellular models could not be elucidated using hNPCs.

Anticonvulsant effects after grafting of rat, porcine, and human mesencephalic neural progenitor cells into the rat subthalamic nucleus.

Cell transplantation based therapy is a promising strategy for treating intractable epilepsies. Inhibition of the subthalamic nucleus (STN) or substantia nigra pars reticulata (SNr) is a powerful experimental approach for remote control of different partial seizure types, when targeting the seizure focus is not amenable. Here, we tested the hypothesis that grafting of embryonic/fetal neural precursor cells (NPCs) from various species (rat, human, pig) into STN or SNr of adult rats induces anticonvulsant effects. To rationally refine this approach, we included NPCs derived from the medial ganglionic eminence (MGE) and ventral mesencephalon (VM), both of which are able to develop a GABAergic phenotype. All VM- and MGE-derived cells showed intense migration behavior after grafting into adult rats, developed characteristics of inhibitory interneurons, and survived at least up to 4 months after transplantation. By using the intravenous pentylenetetrazole (PTZ) seizure threshold test in adult rats, transient anticonvulsant effects were observed after bilateral grafting of NPCs derived from human and porcine VM into STN, but not after SNr injection (site-specificity). In contrast, MGE-derived NPCs did not cause anticonvulsant effects after grafting into STN or SNr (cell-specificity). Neither induction of status epilepticus by lithium-pilocarpine to induce neuronal damage prior to the PTZ test nor pretreatment of MGE cells with retinoic acid and potassium chloride to increase differentiation into GABAergic neurons could enhance anticonvulsant effectiveness of MGE cells. This is the first proof-of-principle study showing anticonvulsant effects by bilateral xenotransplantation of NPCs into the STN. Our study highlights the value of VM-derived NPCs for interneuron-based cell grafting targeting the STN.

Oxidative Damage Control in a Human (Mini-) Organ: Nrf2 Activation Protects against Oxidative Stress-Induced Hair Growth Inhibition.

The in situ control of redox insult in human organs is of major clinical relevance, yet remains incompletely understood. Activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the "master regulator" of genes controlling cellular redox homeostasis, is advocated as a therapeutic strategy for diseases with severely impaired redox balance. It remains to be shown whether this strategy is effective in human organs, rather than only in isolated human cell types. We have therefore explored the role of Nrf2 in a uniquely accessible human (mini-) organ: scalp hair follicles. Microarray and qRT-PCR analysis of human hair follicles after Nrf2 activation using sulforaphane identified the modulation of phase II metabolism, reactive oxygen species clearance, the pentose phosphate pathway, and glutathione homeostasis. Nrf2 knockdown (small interfering RNA) in cultured human hair follicles confirmed the regulation of key Nrf2 target genes (i.e., heme oxygenase-1, NAD(P)H dehydrogenase, quinone 1, glutathione reductase, glutamate-cysteine ligase catalytic subunit, ABCC1, peroxiredoxin 1). Importantly, Nrf2 activation significantly reduced reactive oxygen species levels and associated lipid peroxidation. Nrf2 preactivation reduced premature catagen and hair growth inhibition induced by oxidative stress (H2O2 or menadione), significantly ameliorated the H2O2-dependent increase in matrix keratinocyte apoptosis and reversed the reactive oxygen species-induced reduction in hair matrix proliferation. This study thus provides direct evidence for the crucial role of Nrf2 in protecting human organ function (i.e., scalp hair follicles) against redox insult.