Axonal autophagic vesicle transport in the rat optic nerve in vivo under normal conditions and during acute axonal degeneration.

Neurons pose a particular challenge to degradative processes like autophagy due to their long and thin processes. Autophagic vesicles (AVs) are formed at the tip of the axon and transported back to the soma. This transport is essential since the final degradation of the vesicular content occurs only close to or in the soma. Here, we established an in vivo live-imaging model in the rat optic nerve using viral vector mediated LC3-labeling and two-photon-microscopy to analyze axonal transport of AVs. Under basal conditions in vivo, 50% of the AVs are moving with a majority of 85% being transported in the retrograde direction. Transport velocity is higher in the retrograde than in the anterograde direction. A crush lesion of the optic nerve results in a rapid breakdown of retrograde axonal transport while the anterograde transport stays intact over several hours. Close to the lesion site, the formation of AVs is upregulated within the first 6 h after crush, but the clearance of AVs and the levels of lysosomal markers in the adjacent axon are reduced. Expression of p150Glued, an adaptor protein of dynein, is significantly reduced after crush lesion. In vitro, fusion and colocalization of the lysosomal marker cathepsin D with AVs are reduced after axotomy. Taken together, we present here the first in vivo analysis of axonal AV transport in the mammalian CNS using live-imaging. We find that axotomy leads to severe defects of retrograde motility and a decreased clearance of AVs via the lysosomal system.

Preclinical evaluation of CRISPR-based therapies for Noonan syndrome caused by deep-intronic LZTR1 variants.

Gene variants in LZTR1 are implicated to cause Noonan syndrome associated with a severe and early-onset hypertrophic cardiomyopathy. Mechanistically, LZTR1 deficiency results in accumulation of RAS GTPases and, as a consequence, in RAS-MAPK signaling hyperactivity, thereby causing the Noonan syndrome-associated phenotype. Despite its epidemiological relevance, pharmacological as well as invasive therapies remain limited. Here, personalized CRISPR-Cas9 gene therapies might offer a novel alternative for a curative treatment in this patient cohort. In this study, by utilizing a patient-specific screening platform based on iPSC-derived cardiomyocytes from two Noonan syndrome patients, we evaluated different clinically translatable therapeutic approaches using small Cas9 orthologs targeting a deep-intronic LZTR1 variant to cure the disease-associated molecular pathology. Despite high editing efficiencies in cardiomyocyte cultures transduced with lentivirus or all-in-one adeno-associated viruses, we observed crucial differences in editing outcomes in proliferative iPSCs vs. non-proliferative cardiomyocytes. While editing in iPSCs rescued the phenotype, the same editing approaches did not robustly restore LZTR1 function in cardiomyocytes, indicating critical differences in the activity of DNA double-strand break repair mechanisms between proliferative and non-proliferative cell types and highlighting the importance of cell type-specific screens for testing CRISPR-Cas9 gene therapies.

Human serum-derived α-synuclein auto-antibodies mediate NMDA receptor-dependent degeneration of CNS neurons.

BACKGROUND: Presence of autoantibodies against α-synuclein (α-syn AAb) in serum of the general population has been widely reported. That such peripheral factors may be involved in central nervous system pathophysiology was demonstrated by detection of immunoglobulins (IgGs) in cerebrospinal fluid and brain of Parkinson's disease (PD) patients. Thus, blood-borne IgGs may reach the brain parenchyma through an impaired blood-brain barrier (BBB). FINDINGS: The present study aims to evaluate the patho-physiological impact of α-syn AAbs on primary brain cells, i.e., on spontaneously active neurons and on astrocytes. Exposure of neuron-astrocyte co-cultures to human serum containing α-syn AAbs mediated a dose-dependent reduction of spontaneous neuronal activity, and subsequent neurodegeneration. Removal specifically of α-syn AAbs from the serum prevented neurotoxicity, while purified, commercial antibodies against α-syn mimicked the neurodegenerative effect. Mechanistically, we found a strong calcium flux into neurons preceding α-syn AAbs-induced cell death, specifically through NMDA receptors. NMDA receptor antagonists prevented neurodegeneration upon treatment with α-syn (auto)antibodies. α-syn (auto)antibodies did not affect astrocyte survival. However, in presence of α-syn, astrocytes reacted to α-syn antibodies by secretion of the chemokine RANTES. CONCLUSION: These findings provide a novel basis to explain how a combination of BBB impairment and infiltration of IgGs targeting synuclein may contribute to neurodegeneration in PD and argue for caution with α-syn immunization therapies for treatment of PD.

S-SCAM is essential for synapse formation.

Synapse formation is critical for the wiring of neural circuits in the developing brain. The synaptic scaffolding protein S-SCAM/MAGI-2 has important roles in the assembly of signaling complexes at post-synaptic densities. However, the role of S-SCAM in establishing the entire synapse is not known. Here, we report significant effects of RNAi-induced S-SCAM knockdown on the number of synapses in early stages of network development in vitro. In vivo knockdown during the first three postnatal weeks reduced the number of dendritic spines in the rat brain neocortex. Knockdown of S-SCAM in cultured hippocampal neurons severely reduced the clustering of both pre- and post-synaptic components. This included synaptic vesicle proteins, pre- and post-synaptic scaffolding proteins, and cell adhesion molecules, suggesting that entire synapses fail to form. Correspondingly, functional and morphological characteristics of developing neurons were affected by reducing S-SCAM protein levels; neurons displayed severely impaired synaptic transmission and reduced dendritic arborization. A next-generation sequencing approach showed normal expression of housekeeping genes but changes in expression levels in 39 synaptic signaling molecules in cultured neurons. These results indicate that S-SCAM mediates the recruitment of all key classes of synaptic molecules during synapse assembly and is critical for the development of neural circuits in the developing brain.

Fibroblast growth factor 9 (FGF9)-mediated neurodegeneration: Implications for progressive multiple sclerosis?

AIMS: Fibroblast growth factor (FGF) signalling is dysregulated in multiple sclerosis (MS) and other neurological and psychiatric conditions, but there is little or no consensus as to how individual FGF family members contribute to disease pathogenesis. Lesion development in MS is associated with increased expression of FGF1, FGF2 and FGF9, all of which modulate remyelination in a variety of experimental settings. However, FGF9 is also selectively upregulated in major depressive disorder (MDD), prompting us to speculate it may also have a direct effect on neuronal function and survival. METHODS: Transcriptional profiling of myelinating cultures treated with FGF1, FGF2 or FGF9 was performed, and the effects of FGF9 on cortical neurons investigated using a combination of transcriptional, electrophysiological and immunofluorescence microscopic techniques. The in vivo effects of FGF9 were explored by stereotactic injection of adeno-associated viral (AAV) vectors encoding either FGF9 or EGFP into the rat motor cortex. RESULTS: Transcriptional profiling of myelinating cultures after FGF9 treatment revealed a distinct neuronal response with a pronounced downregulation of gene networks associated with axonal transport and synaptic function. In cortical neuronal cultures, FGF9 also rapidly downregulated expression of genes associated with synaptic function. This was associated with a complete block in the development of photo-inducible spiking activity, as demonstrated using multi-electrode recordings of channel rhodopsin-transfected rat cortical neurons in vitro and, ultimately, neuronal cell death. Overexpression of FGF9 in vivo resulted in rapid loss of neurons and subsequent development of chronic grey matter lesions with neuroaxonal reduction and ensuing myelin loss. CONCLUSIONS: These observations identify overexpression of FGF9 as a mechanism by which neuroaxonal pathology could develop independently of immune-mediated demyelination in MS. We suggest targeting neuronal FGF9-dependent pathways may provide a novel strategy to slow if not halt neuroaxonal atrophy and loss in MS, MDD and potentially other neurodegenerative diseases.

The holo beta-lactoglobulin lozenge reduces symptoms in cat allergy-Evaluation in an allergen exposure chamber and by titrated nasal allergen challenge.

BACKGROUND: The allergists´ tool box in cat allergy management is limited. Clinical studies have shown that holo beta-lactoglobulin (holoBLG) can restore micronutritional deficits in atopic immune cells and alleviate allergic symptoms in a completely allergen-nonspecific manner. With this study, we aimed to provide proof of principle in cat allergy. METHODS: A novel challenge protocol for cat allergy in a standardized ECARF allergen exposure chamber (AEC) was developed. In an open pilot study (NCT05455749), patients with clinically relevant cat allergy were provoked with cat allergen for 120 min in the AEC before and after a 3-month intervention phase (holoBLG lozenge 2x daily). Nasal, conjunctival, bronchial, and pruritus symptoms were scored every 10 min- constituting the total symptom score (TSS). Peak nasal inspiratory flow (PNIF) was measured every 30 min. In addition, a titrated nasal provocation test (NPT) was performed before and after the intervention. Primary endpoint was change in TSS at the end of final exposure compared to baseline. Secondary endpoints included changes in PNIF, NPT, and occurrence of late reactions up to 24 h after exposure. RESULTS: 35 patients (mean age: 40 years) completed the study. Compared to baseline, holoBLG supplementation resulted in significant improvement in median TSS of 50% (p < 0.001), as well as in median nasal flow by 20 L/min (p = 0.0035). 20% of patients reported late reactions after baseline exposure, but 0% after the final exposure. CONCLUSIONS: Cat allergic patients profited from targeted micronutrition with the holoBLG lozenge. As previously seen in other allergies, holoBLG supplementation also induced immune resilience in cat allergies, resulting in significant symptom amelioration.

α-Synuclein Impacts on Intrinsic Neuronal Network Activity Through Reduced Levels of Cyclic AMP and Diminished Numbers of Active Presynaptic Terminals.

α-synuclein (α-Syn) is intimately linked to synucleinopathies like Parkinson's disease and dementia with Lewy bodies. However, the pathophysiological mechanisms that are triggered by this protein are still largely enigmatic. α-Syn overabundance may cause neurodegeneration through protein accumulation and mitochondrial deterioration but may also result in pathomechanisms independent from neuronal cell death. One such proposed pathological mechanism is the influence of α-Syn on non-stimulated, intrinsic brain activity. This activity is responsible for more than 90% of the brain's energyconsumption, and is thus thought to play an eminent role in basic brain functionality. Here we report that α-Syn substantially disrupts intrinsic neuronal network burst activity in a long-term neuronal cell culture model. Mechanistically, the impairment of network activity originates from reduced levels of cyclic AMP and cyclic AMP-mediated signaling as well as from diminished numbers of active presynaptic terminals. The profound reduction of network activity due to α-Syn was mediated only by intracellularly expressed α-Syn, but not by α-Syn that is naturally released by neurons. Conversely, extracellular pre-formed fibrils of α-Syn mimicked the effect of intracellular α-Syn, suggesting that they trigger an off-target mechanism that is not activated by naturally released α-Syn. A simulation-based model of the network activity in our cultures demonstrated that even subtle effect sizes in reducing outbound connectivity, i.e., loss of active synapses, can cause substantial global reductions in non-stimulated network activity. These results suggest that even low-level loss of synaptic output capabilities caused by α-Syn may result in significant functional impairments in terms of intrinsic neuronal network activity. Provided that our model holds true for the human brain, then α-Syn may cause significant functional lesions independent from neurodegeneration.

The relevance of synuclein autoantibodies as a biomarker for Parkinson's disease.

Several studies have investigated if the levels of α-synuclein autoantibodies (α-syn AAb) differ in serum of Parkinson's disease (PD) patients and healthy subjects. Reproducible differences in their levels could serve as a biomarker for PD. The results of previous studies however remain inconclusive. With the largest sample size examined so far, we aimed to validate serum α-syn AAb levels as a biomarker for PD and investigated the presence of AAbs against other synucleins. We performed ELISA and immunoblots to determine synuclein AAb levels in the serum of 295 subjects comprising 157 PD patients from two independent cohorts, 46 healthy subjects, and 92 patients with other neurodegenerative disorders. Although serum α- and ß-syn AAb levels were significantly reduced in patients with PD and other neurodegenerative disorders as compared to controls, the AAb levels displayed high inter-and intra-cohort variability. Furthermore, α-syn AAb levels showed no correlation to clinical parameters like age, disease duration, disease severity, and gender, that might also be directed against beta- and gamma-syn. In conclusion, serum synuclein AAb levels do allow the separation of PD from healthy subjects but not from other neurodegenerative disorders. Thus, synuclein AAbs cannot be regarded as a reliable biomarker for PD.

Optimized pharmacological control over the AAV-Gene-Switch vector for regulable gene therapy.

Gene therapy in its current design is an irreversible process. It cannot be stopped in case of unwanted side effects, nor can expression levels of therapeutics be adjusted to individual patient's needs. Thus, the Gene-Switch (GS) system for pharmacologically regulable neurotrophic factor expression was established for treatment of parkinsonian patients. Mifepristone, the synthetic steroid used to control transgene expression of the GS vector, is an approved clinical drug. However, pharmacokinetics and -dynamics of mifepristone vary considerably between different experimental animal species and depend on age and gender. In humans, but not in any other species, mifepristone binds to a high-affinity plasma carrier protein. We now demonstrate that the formulation of mifepristone can have robust impact on its ability to activate the GS system. Furthermore, we show that a pharmacological booster, ritonavir (Rtv), robustly enhances the pharmacological effect of mifepristone, and allows it to overcome gender- and species-specific pharmacokinetic and -dynamic issues. Most importantly, we demonstrate that the GS vector can be efficiently controlled by mifepristone in the presence of its human plasma carrier protein, α1-acid glycoprotein, in a "humanized" rat model. Thus, we have substantially improved the applicability of the GS vector toward therapeutic use in patients.

Face masks suitable for preventing COVID-19 and pollen allergy. A study in the exposure chamber.

BACKGROUND: Since the outbreak of the coronavirus pandemic, the population in Germany has been asked to wear face masks in public areas. The masks are accepted by the public. People with a pollen allergy have an interest in knowing whether masks can also provide protection against pollen and thus prevent symptoms even without medication. METHOD: In order to evaluate the potential 'antipollen effect' of face masks, 14 adults with confirmed grass pollen-induced allergic rhinoconjunctivitis were exposed to grass pollen for a period of two hours following a standardised protocol. The test was conducted outside of the grass pollen season. The subjects wore either no mask, a medical mask or a FFP2 mask. RESULTS: Subjects wearing either mask were clearly able to avoid both nasal and conjunctival symptoms. There were no significant differences between the two masks in terms of effect. Mask wearing to prevent pollen exposure clearly supports overall well-being. CONCLUSION: Wearing a mask during pollen season can be recommended as an effective nondrug option for people with a pollen allergy. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s40629-021-00180-8) contains supplementary material, which is available to authorized users.

Dementia with Lewy bodies-associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions.

Beta (ß)-synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson's disease-related alpha (α)-synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in central nervous system (CNS) neurons in vitro and in vivo, albeit at a slower pace as compared with α-Syn. Here, we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of dementia with Lewy bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but it has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but it does not aggravate neurodegeneration. ß-Syn wild type (WT), V70M and P123H formed proteinase K-resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared with α-Syn. Under cell-free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared with WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, which are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn and is thus likely to be directly involved into the etiology of DLB. Overall, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.

Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Syntenin-knock out reduces exosome turnover and viral transduction.

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery.

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood-brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

Dopamine promotes the neurodegenerative potential of ß-synuclein.

A contribution of α-Synuclein (α-Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related ß-Synuclein (ß-Syn) on these disorders remains enigmatic. ß-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD ß-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of ß-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced ß-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with ß-Syn, thereby enabling structural and functional modifications. Interaction of DCH with ß-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric ß-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and ß-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that ß-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.

First evaluation of a symbiotic food supplement in an allergen exposure chamber in birch pollen allergic patients.

BACKGROUND: Allergic rhinitis/rhinoconjunctivitis is the most common immune disease worldwide, but still largely underestimated, underdiagnosed, and undertreated. Dysbiosis and reduced microbial diversity is linked to the development of allergies, and the immunomodulatory effects of pro- and prebiotics might be used to counteract microbiome dysbiosis in allergy. Adequate symbiotic (multi-strain pro-, plus prebiotic) supplementation can be suggested as a complementary approach in the management of allergic rhinitis. OBJECTIVE: The effects of the daily intake of a symbiotic food supplement (combination of Lactobacillus acidophilus NCFM and Bifidobacterium lactis BL-04 with Fructo-Oligosaccharides) for 4 months in birch pollen allergic rhinoconjunctivitis patients were investigated for the first time in an allergen exposure chamber (AEC) allowing standardised, reproducible pollen exposure before and after intake. METHODS: Eligible patients were exposed to birch pollen (8000 pollen/m³ for 120 min) at the GA2LEN AEC, at baseline (V1) and final visit (V3) outside the season. The Total Symptom Score (TSS) and the scores for nose, eye, bronchial system, and others were evaluated every 10 min during exposure. Other secondary endpoints were the changes in well-being, Peak Nasal Inspiratory Flow (PNIF), lung function parameters, and safety. Co-primary endpoints were differences in Total Nasal Symptom Score (TNSS) and TSS after 120 min of exposure between both visits. Temporal evolution of symptom scores were analysed in an exploratory way using linear mixed effects models. RESULTS: 27 patients (mean age 45 years, 15% male) completed the study. Both co-primary endpoints showed significant improvement after intake of the symbiotic. Median TNSS and TSS were decreased 50% and 80% at 120 min (adjusted p-value = 0.025 and p < 0.01 respectively).All four symptom scores and the personal well-being, improved to a clinically relevant extent over time, visible by a weaker increase in symptoms during 120 min of the final birch pollen exposure. No relevant differences were observed for PNIF, PEF, and spirometry. There were no airway obstructions or lung restrictions before and after both exposures. Late phase reactions after exposure were reduced after V3, documenting a better birch pollen tolerability of the patients. The safety and tolerability profile of the symbiotic food supplement was excellent, no adverse events (AEs) were observed. CONCLUSIONS: This first evaluation of a symbiotic food supplement in an AEC in rhinoconjunctivitis patients with or without asthma induced by birch pollen revealed a significant beneficial effect, harnessing significant improvements of symptoms and well-being while maintaining an excellent safety and tolerability profile.

Overshooting Subcellular Redox-Responses in Rett-Mouse Hippocampus during Neurotransmitter Stimulation.

Rett syndrome (RTT) is a neurodevelopmental disorder associated with disturbed neuronal responsiveness and impaired neuronal network function. Furthermore, mitochondrial alterations and a weakened cellular redox-homeostasis are considered part of the complex pathogenesis. So far, overshooting redox-responses of MeCP2-deficient neurons were observed during oxidant-mediated stress, hypoxia and mitochondrial inhibition. To further clarify the relevance of the fragile redox-balance for the neuronal (dys)function in RTT, we addressed more physiological stimuli and quantified the subcellular redox responses to neurotransmitter-stimulation. The roGFP redox sensor was expressed in either the cytosol or the mitochondrial matrix of cultured mouse hippocampal neurons, and the responses to transient stimulation by glutamate, serotonin, dopamine and norepinephrine were characterized. Each neurotransmitter evoked more intense oxidizing responses in the cytosol of MeCP2-deficient than in wildtype neurons. In the mitochondrial matrix the neurotransmitter-evoked oxidizing changes were more moderate and more uniform among genotypes. This identifies the cytosol as an important reactive oxygen species (ROS) source and as less stably redox buffered. Fura-2 imaging and extracellular Ca2+ withdrawal confirmed cytosolic Ca2+ transients as a contributing factor of neurotransmitter-induced redox responses and their potentiation in the cytosol of MeCP2-deficient neurons. Chemical uncoupling demonstrated the involvement of mitochondria. Nevertheless, cytosolic NADPH- and xanthine oxidases interact to play the leading role in the neurotransmitter-mediated oxidizing responses. As exaggerated redox-responses were already evident in neonatal MeCP2-deficient neurons, they may contribute remarkably to the altered neuronal network performance and the disturbed neuronal signaling, which are among the hallmarks of RTT.

Neuronal Trans-differentiation by Transcription Factors Ascl1 and Nurr1: Induction of a Dopaminergic Neurotransmitter Phenotype in Cortical GABAergic Neurons.

Neurons with a desired neurotransmitter phenotype can be differentiated from induced pluripotent stem cells or from somatic cells only through tedious protocols with relatively low yield. Readily available cortical neurons isolated from embryonic rat brain, which have already undergone a complete neuronal differentiation process, might serve as alternative template source. These cultures consist of 85% glutamatergic and 15% GABAergic neurons, and we attempted to trans-differentiate them into dopaminergic neurons. Transcription factors Nurr1, Lmx1A and Pitx3, essential determinants of a dopaminergic cell fate during CNS development, were not sufficient to induce tyrosine hydroxylase expression in a significant number of cells. Combining Nurr1 with the generic neuronal differentiator and re-programming factor Ascl1, however, resulted in generation of neurons which express dopaminergic markers TH, AADC, VMAT2 and DAT. Only neurons of GABAergic phenotype could be trans-differentiated towards a dopaminergic neurotransmitter phenotype, while for glutamatergic neurons, this process proved to be neurotoxic. Intriguingly, GABAergic neurons isolated from embryonal midbrain could not be trans-differentiated into dopaminergic neurons by Ascl1 and Nurr1. Thus, in principle, post-mitotic embryonal neurons can serve as templates for neurons with a desired neurotransmitter phenotype. However, neurotransmitter phenotype plasticity critically depends on the differentiation history of the template neurons, which can result in relatively low yields of dopaminergic neurons.