Alteration of Mitochondrial Integrity as Upstream Event in the Pathophysiology of SOD1-ALS.

Little is known about the early pathogenic events by which mutant superoxide dismutase 1 (SOD1) causes amyotrophic lateral sclerosis (ALS). This lack of mechanistic understanding is a major barrier to the development and evaluation of efficient therapies. Although protein aggregation is known to be involved, it is not understood how mutant SOD1 causes degeneration of motoneurons (MNs). Previous research has relied heavily on the overexpression of mutant SOD1, but the clinical relevance of SOD1 overexpression models remains questionable. We used a human induced pluripotent stem cell (iPSC) model of spinal MNs and three different endogenous ALS-associated SOD1 mutations (D90Ahom, R115Ghet or A4Vhet) to investigate early cellular disturbances in MNs. Although enhanced misfolding and aggregation of SOD1 was induced by proteasome inhibition, it was not affected by activation of the stress granule pathway. Interestingly, we identified loss of mitochondrial, but not lysosomal, integrity as the earliest common pathological phenotype, which preceded elevated levels of insoluble, aggregated SOD1. A super-elongated mitochondrial morphology with impaired inner mitochondrial membrane potential was a unifying feature in mutant SOD1 iPSC-derived MNs. Impaired mitochondrial integrity was most prominent in mutant D90Ahom MNs, whereas both soluble disordered and detergent-resistant misfolded SOD1 was more prominent in R115Ghet and A4Vhet mutant lines. Taking advantage of patient-specific models of SOD1-ALS in vitro, our data suggest that mitochondrial dysfunction is one of the first crucial steps in the pathogenic cascade that leads to SOD1-ALS and also highlights the need for individualized medical approaches for SOD1-ALS.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

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.

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.

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.

Transcriptome-wide Profiling of Cerebral Cavernous Malformations Patients Reveal Important Long noncoding RNA molecular signatures.

Cerebral cavernous malformations (CCMs) are low-flow vascular malformations in the brain associated with recurrent hemorrhage and seizures. The current treatment of CCMs relies solely on surgical intervention. Henceforth, alternative non-invasive therapies are urgently needed to help prevent subsequent hemorrhagic episodes. Long non-coding RNAs (lncRNAs) belong to the class of non-coding RNAs and are known to regulate gene transcription and involved in chromatin remodeling via various mechanism. Despite accumulating evidence demonstrating the role of lncRNAs in cerebrovascular disorders, their identification in CCMs pathology remains unknown. The objective of the current study was to identify lncRNAs associated with CCMs pathogenesis using patient cohorts having 10 CCM patients and 4 controls from brain. Executing next generation sequencing, we performed whole transcriptome sequencing (RNA-seq) analysis and identified 1,967 lncRNAs and 4,928 protein coding genes (PCGs) to be differentially expressed in CCMs patients. Among these, we selected top 6 differentially expressed lncRNAs each having significant correlative expression with more than 100 differentially expressed PCGs. The differential expression status of the top lncRNAs, SMIM25 and LBX2-AS1 in CCMs was further confirmed by qRT-PCR analysis. Additionally, gene set enrichment analysis of correlated PCGs revealed critical pathways related to vascular signaling and important biological processes relevant to CCMs pathophysiology. Here, by transcriptome-wide approach we demonstrate that lncRNAs are prevalent in CCMs disease and are likely to play critical roles in regulating important signaling pathways involved in the disease progression. We believe, that detailed future investigations on this set of identified lncRNAs can provide useful insights into the biology and, ultimately, contribute in preventing this debilitating disease.

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.

FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

Modeling Parkinson's disease in midbrain-like organoids.

Modeling Parkinson's disease (PD) using advanced experimental in vitro models is a powerful tool to study disease mechanisms and to elucidate unexplored aspects of this neurodegenerative disorder. Here, we demonstrate that three-dimensional (3D) differentiation of expandable midbrain floor plate neural progenitor cells (mfNPCs) leads to organoids that resemble key features of the human midbrain. These organoids are composed of midbrain dopaminergic neurons (mDANs), which produce and secrete dopamine. Midbrain-specific organoids derived from PD patients carrying the LRRK2-G2019S mutation recapitulate disease-relevant phenotypes. Automated high-content image analysis shows a decrease in the number and complexity of mDANs in LRRK2-G2019S compared to control organoids. The floor plate marker FOXA2, required for mDAN generation, increases in PD patient-derived midbrain organoids, suggesting a neurodevelopmental defect in mDANs expressing LRRK2-G2019S. Thus, we provide a robust method to reproducibly generate 3D human midbrain organoids containing mDANs to investigate PD-relevant patho-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.

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization.

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function. SIGNIFICANCE STATEMENT: Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.