Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE

A Balanced Translocation in Kallmann Syndrome Implicates a Long Noncoding RNA, RMST, as a GnRH Neuronal Regulator.

CONTEXT: Kallmann syndrome (KS) is a rare, genetically heterogeneous Mendelian disorder. Structural defects in KS patients have helped define the genetic architecture of gonadotropin-releasing hormone (GnRH) neuronal development in this condition. OBJECTIVE: Examine the functional role a novel structural defect affecting a long noncoding RNA (lncRNA), RMST, found in a KS patient. DESIGN: Whole genome sequencing, induced pluripotent stem cells and derived neural crest cells (NCC) from the KS patient were contrasted with controls. SETTING: The Harvard Reproductive Sciences Center, Massachusetts General Hospital Center for Genomic Medicine, and Singapore Genome Institute. PATIENT: A KS patient with a unique translocation, t(7;12)(q22;q24). INTERVENTIONS/MAIN OUTCOME MEASURE/RESULTS: A novel translocation was detected affecting the lncRNA, RMST, on chromosome 12 in the absence of any other KS mutations. Compared with controls, the patient's induced pluripotent stem cells and NCC provided functional information regarding RMST. Whereas RMST expression increased during NCC differentiation in controls, it was substantially reduced in the KS patient's NCC coincident with abrogated NCC morphological development and abnormal expression of several "downstream" genes essential for GnRH ontogeny (SOX2, PAX3, CHD7, TUBB3, and MKRN3). Additionally, an intronic single nucleotide polymorphism in RMST was significantly implicated in a genome-wide association study associated with age of menarche. CONCLUSIONS: A novel deletion in RMST implicates the loss of function of a lncRNA as a unique cause of KS and suggests it plays a critical role in the ontogeny of GnRH neurons and puberty

Dysfunction of spatacsin leads to axonal pathology in SPG11-linked hereditary spastic paraplegia

Hereditary spastic paraplegias are a group of inherited motor neuron diseases characterized by progressive paraparesis and spasticity. Mutations in the spastic paraplegia gene SPG11, encoding spatacsin, cause an autosomal-recessive disease trait; however, the precise knowledge about the role of spatacsin in neurons is very limited. We for the first time analyzed the expression and function of spatacsin in human forebrain neurons derived from human pluripotent stem cells including lines from two SPG11 patients and two controls. SPG11 patients'-derived neurons exhibited downregulation of specific axonal-related genes, decreased neurite complexity and accumulation of membranous bodies within axonal processes. Altogether, these data point towards axonal pathologies in human neurons with SPG11 mutations. To further corroborate spatacsin function, we investigated human pluripotent stem cell-derived neurons and mouse cortical neurons. In these cells, spatacsin was located in axons and dendrites. It colocalized with cytoskeletal and synaptic vesicle (SV) markers and was present in synaptosomes. Knockdown of spatacsin in mouse cortical neurons evidenced that the loss of function of spatacsin leads to axonal instability by downregulation of acetylated tubulin. Finally, time-lapse assays performed in SPG11 patients'-derived neurons and spatacsin-silenced mouse neurons highlighted a reduction in the anterograde vesicle trafficking indicative of impaired axonal transport. By employing SPG11 patient-derived forebrain neurons and mouse cortical neurons, this study provides the first evidence that SPG11 is implicated in axonal maintenance and cargo trafficking. Understanding the cellular functions of spatacsin will allow deciphering mechanisms of motor cortex dysfunction in autosomal-recessive hereditary spastic paraplegia

Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients’ neurons

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC− toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca2+ influx promoted by molecules locked in a Ca2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux

Die Etablierung eines Modells der SPG4 betreffenden Heriditären Spastischen Paraplegie basierend auf der Nutzung von humanen induzierten pluripotenten Stammzellen

The hereditary spastic paraplegias (HSP) are a heterogeneous group of motor neuron diseases, characterized by progressive spasticity and paresis of the lower limbs. Research in the past two decades focused on screening for genes and gene loci associated with HSP, identifying the huge cohort of 74 gene loci that are disease causative. Mutations in SPG4 (spastic paraplegia 4; also known as SPAST), encoding Spastin, are the most frequent cause of HSP, accounting for 40-50% of all autosomal dominant cases. Animal models for SPG4-related HSP have been generated in Drosophila, zebrafish and mouse. They have contributed to our current knowledge on the role of Spastin in microtubule dynamics and were used to generate hypotheses for the potential pathological processes that might underlie HSP in humans. However, these animal models have generated inconsistent cellular phenotypes across the different species and vary largely in the degree of motor deficits they develop, thereby not recapitulating some major aspects of HSP seen in human patients. To understand, how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation in SPG4, and from two age matched controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiencies and mature functionally in vitro. We show here, that neurons from SPG4 patients had a comparably decreased total neurite length and arborization, indicating an altered neurite complexity. In addition, numerous neurite swellings with severely disrupted microtubule structures were identified in SPG4 neurons. The neurite swellings and the reduced neurite complexity were associated with reduced levels of all known Spastin isoforms in SPG4 neurons. These phenotypes were fully reverted by lentiviral overexpression of either M1 or M87 Spastin. Neurite swellings in SPG4 neurons partly accumulated Tubulin with microtubule stabilizing posttranslational modifications like acetylation and detyrosination. Nevertheless, the total amount of acetylated and detyrosinated Tubulin within the cells was unaltered. In line with this, an endogenous upregulation of p60 Katanin was detected in patient cells. P60 Katanin is known to have an important role in regulating microtubule dynamics within neurons, and therefore might partly compensate for the loss of Spastin. Furthermore, a prominent alteration of the movement of mitochondria towards less retrograde transport was observed in SPG4 neurons and might contribute to HSP pathogenesis. Interestingly, we identified a new Spastin isoform, which is generated by exon 15 skipping. We were able to detect this isoform in human post mortem spinal cord tissue, suggesting the physiological expression of this isoform exists in vivo. In conclusion, our data are consistent with a gene-dosage dependent neurodegenerative effect of Spastin in human SPG4 neurons and suggest that bringing wild-type Spastin expression back to normal levels might be an opportunity to halt neuronal degeneration in patients. Our human in vitro model provides the foundation for compound screenings with the goal to restore physiological Spastin levels in patient samples and thereby paves the road to discover new treatment strategies for SPG4 related HSP.Die Hereditäre Spastische Paraplegie (HSP) ist eine heterogene Gruppe von Motoneuronerkrankungen, die durch eine progressive Spastik und Parese der unteren Extremitäten charakterisiert sind. Das Hauptaugenmerk der HSP Forschung der vergangenen 20 Jahre lag auf der Identifikation zahlreicher Gene und Genloci, die mit HSP assoziiert sind. Nach dem aktuellen Stand (30.03.2014) sind bereits 74 Genloci bekannt, die zu einer Form der HSP führen können. Mutationen im SPG4 Gen (engl. spastic parplegia 4; auch als SPAST bekannt), welches das Protein Spastin kodiert, sind die häufigste Ursache für eine HSP und verursachen etwa 40-50% aller autosomal dominant vererbten Fälle. Tiermodelle der SPG4 wurden in Drosophila, Zebrafisch und der Maus generiert. Sie haben zu unserem derzeitigen Verständnis über die Rolle von Spastin in der Regulation der Dynamik der Mikrotubuli beigetragen. Desweiteren wurden sie genutzt, um Hypothesen über die potentiellen Pathomechanismen zu generieren, die zur Erkrankung im Menschen führen könnten. Jedoch weisen diese Tiermodelle inkonsistente Phänotypen sowohl auf zellulärer Ebene, als auch im Umfang der Einschränkung der Motorik auf. Demnach können sie nur limitiert wesentliche Aspekte der humanen Pathogenese rekapitulieren, die bei HSP Patienten gefunden werden. Um zu analysieren, welchen Einfluß SPG4 Mutationen auf menschliche Neuronen haben, generierten wir humane induzierte pluripotente Stamzellen (hiPSZ) sowohl von zwei Patienten mit einer c.1684C>T nonsense Mutation im SPG4 Gen, als auch von zwei gesunden, auf das Alter der Patienten abgestimmten Kontrollen. Sowohl die SPG4, als auch die Kontroll hiPSZ Linien konnten mit vergleichbarer Effizienz zu Neuronen und Gliazellen differenziert werden, welche auch in vitro funktional ausreiften. Wir zeigen, dass SPG4 Neurone eine verkürzte Neuritenlänge und einen reduzierten Verzweigungsgrad aufweisen. Diese Befunde deuten auf eine reduzierte Neuritenkomplexizität in den SPG4 Neuronen hin. Desweitern wurden zahlreiche Neuritenschwellungen mit prominenten Unterbrechungen der Mikrotubulibündel in den SPG4 Neuronen identifiziert. Wir konnten die Neuritenschwellungen, als auch die reduzierte Neuritenkomplexizität auf eine Reduktion der bekannten Spastinisoformen zurückführen, da eine lentivirale Überexpression der M1 oder M87 Isoform von Spastin die Phänotypen in den SPG4 Zellen revidiert hat. Die Neuritenschwellungen akkumulierten teilweise Tubulinproteine, die posttranslationale Modifikationen wie Acetylierung und Detyrosinierung trugen. Diese Modifkationen sind charakteristisch für stabilisierte Mikrotubulibündel. Nichtsdestotrotz, konnten wir keine Veränderung in der Gesamtmenge von acetyliertem oder detyrosiniertem Tubulin in diesen Zellen feststellen. Im Einklang hiermit, konnten wir eine endogene Hochregulation von p60 Katanin feststellen. P60 Katanin ist ein Protein, dass bereits mit der Regulation der Dynamik der Mikrotubuli in neuronalen Zellen assoziiert wurde und könnte somit den Verlust von Spastin in SPG4 Neuronen teilweise kompensieren. Zusätzlich konnten wir in SPG4 Neuronen eine auffällige Veränderung der aktiven axonalen Verteilung der Mitochondrien nachweisen, wobei im Speziellen weniger Mitochondrien retrograd transportiert wurden. Diese Veränderung im axonalen Transport könnte mit der Pathogenese der HSP assoziiert sein. Erstaunlicherweise, waren wir in der Lage eine neue Spastinisoform zu identifizieren, bei der Exon 15 alternativ gespleißt wird. Diese Isoform konnten wir auch in humanem post-mortem Gewebe des Rückenmarks nachweisen, was darauf hindeuten könnte, dass diese Isoform auch physiologisch in vivo existiert. Zusammenfassend weisen unsere Daten auf einen Gendosis abhängigen, neurodegenerativen Effekt der Spastinmenge in humanen SPG4 Neuronen hin. Darüberhinaus deuten die Daten darauf hin, dass eine Erhöhung der Spastinmenge in SPG4 Patienten eine Möglichkeit darstellen könnte, die Degeneration der Nervenzellen in den Patienten aufzuhalten. Das hier enwickelte humane in vitro Modell der HSP bildet die Grundlage für das Screening von Substanzen mit dem Ziel, die Spastinmenge auf physiologisches Niveau zu erhöhen und ebnet somit den Weg für die Entwicklung neuer therapeutischer Ansätze für die SPG4 assoziierte HSP

387. Gene Delivery of APOE2 Reduces Amyloid Pathology in Transgenic Mouse Models of Alzheimer\u27s Disease

The deposition of amyloid β-peptides (Aβ), cleavage products of the amyloid precursor protein (APP) by β- and γ-secretases, in brain represents a pathological hallmark of Alzheimer\u27s disease (AD). Apolipoprotein E (APOE) ε4 allele carriers have an increased risk to develop AD and an earlier age of onset, whereas carriers of the ε2 allele have reduced risk and a delayed age of onset. APOE is also a major determinant of brain Aβ and amyloid burden in humans and in several transgenic mouse models of AD (E4\u3eE3\u3eE2). We have previously reported that lentivirus-mediated intraparenchymal gene delivery of APOE2 significantly reduces brain Aβ levels and amyloid plaque burden in PDAPP mice. To extend these findings, we administered an rh.10 serotype adeno-associated viral vector expressing the gene (AAVrh.10-APOE2, 1.0X1010 viral genomes (vg)) directly into the hippocampus of 9-month-old PDAPP mice, a mouse model of AD-related amyloidosis. Eight weeks post-injection, AAVrh.10-APOE2 administration resulted in 5-6 times higher levels of APOE2 expression than targeted replacement (wild-type) mice and a marked decrease in both soluble (~33% reduction. P\u3c0.05) and insoluble Aβ42 levels (~70% reduction. P\u3c0.001) compared to control mice. Given the important role of APOE4 in AD risk and amyloid burden, we next assessed how gene delivery of APOE2 affects amyloid pathology in APP.PS1/TRE4 mice where brain Aβ/amyloid deposition is dependent on APOE4 expression. AAVrh.10-APOE2 (0.25X1010, 0.5X1010, or 1×1010 vg) was bilaterally administered into the hippocampus of 2.5-month-old APP.PS1/TRE4 mice. Eight weeks post-injection, there was a dose-dependent increase in APOE expression and a corresponding dose-dependent decrease in insoluble and soluble Aβ levels in the hippocampus of mice treated with AAVrh.10-APOE2, suggesting that overexpression of APOE2 effectively counteracts the detrimental effects of APOE4 on amyloid pathology. We also investigated the effects of AAVrh.10-APOE2 treatment on various proteins associated with Aβ production and clearance by Western analysis. No significant differences were observed in the relative hippocampal levels of APP, β-secretase, C99 (APP cleavage product of β-secretase), or C83 (a non-amyloidogenic APP cleavage product by α-secretase) between mice treated with AAVrh.10-APOE2 or a control vector, suggesting no effect on Aβ production. By contrast, the levels of insulin-degrading enzyme (IDE, an Aβ-degrading enzyme) and ATG5/LC3 (the signaling pathway responsible for autophagy) were significantly (P\u3c0.001 and P\u3c0.05 respectively) elevated in the hippocampus of AAVrh.10-APOE2-treated mice. Taken together, the data demonstrates that AAVrh.10-mediated delivery of APOE2 effectively reduces Aβ pathology in the hippocampus of APP mutant mice expressing either murine Apoe or APOE4. The latter may be due to an enhancement of Aβ metabolism or clearance. Gene delivery of APOE2 may represent a potential therapeutic strategy for treating or preventing AD

Latent Toxoplasma

Background. Human immunodeficiency virus (HIV)–associated neurocognitive disorders persist despite suppressive antiretroviral therapy (ART). Because latent Toxoplasma infection (LTI) may adversely impact brain function, we investigated its impact on neurocognitive impairment (NCI) in people living with HIV disease. Methods. Two hundred sixty-three HIV-infected adults underwent comprehensive neurocognitive assessments and had anti-Toxoplasma gondii immunoglobulin G (anti-Toxo IgG) measured by qualitative and quantitative enzyme-linked immunosorbent assays. Results. Participants were mostly middle-aged white men who were taking ART (70%). LTI was detected in 30 (11.4%) participants and was associated with a significantly greater prevalence of global NCI (LTI positive [LTI(+)] = 57% and LTI negative [LTI(–)] = 34%) (odds ratio, 1.67; 95% confidence interval, 1.17–2.40; P = .017). Deficits were more prevalent in the LTI(+) vs the LTI(–) group in 6 of 7 cognitive domains with statistical significance reached for delayed recall (P < .01). The probability of NCI increased with higher CD4(+) T-cell counts among LTI(+) individuals but with lower CD4(+) T-cell counts in LTI(–) persons. A strong correlation (r = .93) between anti-Toxo IgG levels and global deficit score was found in a subgroup of 9 patients. Biomarkers indicative of central nervous system inflammation did not differ between LTI(+) and LTI(–) participants. Conclusions. In this cross-sectional analysis, LTI was associated with NCI, especially in those with higher CD4(+) T-cell counts. Longitudinal studies to investigate the role of neuroinflammation and neuronal injury in LTI patients with NCI and trials of anti-Toxoplasma therapy should be pursued