Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features.

Autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA receptors have been identified in 20%-25% of patients with frontotemporal lobar degeneration (FTLD). Data from patients and in vitro/ex vivo pre-clinical studies indicate that anti-GluA3 hIgGs negatively affect glutamatergic neurotransmission. However, whether and how the chronic presence of anti-GluA3 hIgGs triggers synaptic dysfunctions and the appearance of FTLD-related neuropathological and behavioural signature has not been clarified yet. To address this question, we developed and characterized a pre-clinical mouse model of passive immunization with anti-GluA3 hIgGs purified from patients. In parallel, we clinically compared FTLD patients who were positive for anti-GluA3 hIgGs to negative ones. Clinical data showed that the presence of anti-GluA3 hIgGs defined a subgroup of patients with distinct clinical features. In the preclinical model, anti-GluA3 hIgGs administration led to accumulation of phospho-tau in the postsynaptic fraction and dendritic spine loss in the prefrontal cortex. Remarkably, the preclinical model exhibited behavioural disturbances that mostly reflected the deficits proper of patients positive for anti-GluA3 hIgGs. Of note, anti-GluA3 hIgGs-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission with a positive allosteric modulator of AMPA receptors. Overall, our study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and pathology and identified a specific subgroup of FTLD patients. Our findings will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs.

C9orf72 hexanucleotide repeat expansion leads to altered neuronal and dendritic spine morphology and synaptic dysfunction.

Frontotemporal lobar degeneration (FTLD) comprises a heterogenous group of progressive neurodegenerative syndromes. To date, no validated biomarkers or effective disease-modifying therapies exist for the different clinical or genetic subtypes of FTLD. The most common genetic cause underlying FTLD and amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion in the C9orf72 gene (C9-HRE). FTLD is accompanied by changes in several neurotransmitter systems, including the glutamatergic, GABAergic, dopaminergic, and serotonergic systems and many clinical symptoms can be explained by disturbances in these systems. Here, we aimed to elucidate the effects of the C9-HRE on synaptic function, molecular composition of synapses, and dendritic spine morphology. We overexpressed the pathological C9-HRE in cultured E18 mouse primary hippocampal neurons and characterized the pathological, morphological, and functional changes by biochemical methods, confocal microscopy, and live cell calcium imaging. The C9-HRE-expressing neurons were confirmed to display the pathological RNA foci and DPR proteins. C9-HRE expression led to significant changes in dendritic spine morphologies, as indicated by decreased number of mushroom-type spines and increased number of stubby and thin spines, as well as diminished neuronal branching. These morphological changes were accompanied by concomitantly enhanced susceptibility of the neurons to glutamate-induced excitotoxicity as well as augmented and prolonged responses to excitatory stimuli by glutamate and depolarizing potassium chloride as compared to control neurons. Mechanistically, the hyperexcitation phenotype in the C9-HRE-expressing neurons was found to be underlain by increased activity of extrasynaptic GluN2B-containing N-methyl-d-aspartate (NMDA) receptors. Our results are in accordance with the idea suggesting that C9-HRE is associated with enhanced excitotoxicity and synaptic dysfunction. Thus, therapeutic interventions targeted to alleviate synaptic disturbances might offer efficient avenues for the treatment of patients with C9-HRE-associated FTLD.

Deficient neurotransmitter systems and synaptic function in frontotemporal lobar degeneration-Insights into disease mechanisms and current therapeutic approaches.

Frontotemporal lobar degeneration (FTLD) comprises a heterogenous group of fatal neurodegenerative diseases and, to date, no validated diagnostic or prognostic biomarkers or effective disease-modifying therapies exist for the different clinical or genetic subtypes of FTLD. Current treatment strategies rely on the off-label use of medications for symptomatic treatment. Changes in several neurotransmitter systems including the glutamatergic, GABAergic, dopaminergic, and serotonergic systems have been reported in FTLD spectrum disease patients. Many FTLD-related clinical and neuropsychiatric symptoms such as aggressive and compulsive behaviour, agitation, as well as altered eating habits and hyperorality can be explained by disturbances in these neurotransmitter systems, suggesting that their targeting might possibly offer new therapeutic options for treating patients with FTLD. This review summarizes the present knowledge on neurotransmitter system deficits and synaptic dysfunction in model systems and patients harbouring the most common genetic causes of FTLD, the hexanucleotide repeat expansion in C9orf72 and mutations in the granulin (GRN) and microtubule-associated protein tau (MAPT) genes. We also describe the current pharmacological treatment options for FLTD that target different neurotransmitter systems.

Diagnosis and Therapy of Female Genital Malformations (Part 1). Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Registry Number 015/052, May 2019).

Objectives Female genital malformations may take the form of individual entities, they may involve neighboring organs or they may occur in the context of complex syndromes. Given the anatomical structures of the vulva, vagina, uterus and adnexa, the clinical picture of malformations may vary greatly. Depending on the extent of the malformation, organs of the urinary system or associated malformations may also be involved. Methods This S2k-guideline was developed by representative members from different medical specialties and professions as part of the guidelines program of the DGGG, SGGG and OEGGG. The recommendations and statements were developed using a structured consensus process with neutral moderation and voted on. Recommendations The guideline is the first comprehensive presentation of the symptoms, diagnosis and treatment options for female genital malformations. Additional chapters on classifications and transition were included.

Diagnosis and Therapy of Female Genital Malformations (Part 2). Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Registry Number 015/052, May 2019).

Objectives Female genital malformations may be present in the form of individual entities, they may involve neighboring organs or they may occur in the context of complex syndromes. Given the anatomical structures of the vulva, vagina, uterus and uterine appendages, the clinical picture of malformations varies greatly. Methods This S2k-guideline was developed by representative members from different medical specialties and professions as part of the guidelines program of the DGGG, SGGG and OEGGG. The recommendations and statements were developed and voted on using a structured consensus process with neutral moderation. Recommendations This guideline is the first comprehensive summary of female genital malformations from infancy to adulthood which covers clinical examinations, diagnostic workups and treatment options. Additional chapters have been included on complex urogenital malformations, vascular malformations, psychosomatic care, and tumor risk.

FTLD Patient-Derived Fibroblasts Show Defective Mitochondrial Function and Accumulation of p62.

Frontotemporal lobar degeneration (FTLD) is a clinically, genetically, and neuropathologically heterogeneous group of neurodegenerative syndromes, leading to progressive cognitive dysfunction and frontal and temporal atrophy. C9orf72 hexanucleotide repeat expansion (C9-HRE) is the most common genetic cause of FTLD, but pathogenic mechanisms underlying FTLD are not fully understood. Here, we compared cellular features and functional properties, especially related to protein degradation pathways and mitochondrial function, of FTLD patient-derived skin fibroblasts from C9-HRE carriers and non-carriers and healthy donors. Fibroblasts from C9-HRE carriers were found to produce RNA foci, but no dipeptide repeat proteins, and they showed unchanged levels of C9orf72 mRNA transcripts. The main protein degradation pathways, the ubiquitin-proteasome system and autophagy, did not show alterations between the fibroblasts from C9-HRE-carrying and non-carrying FTLD patients and compared to healthy controls. An increase in the number and size of p62-positive puncta was evident in fibroblasts from both C9-HRE carriers and non-carriers. In addition, several parameters of mitochondrial function, namely, basal and maximal respiration and respiration linked to ATP production, were significantly reduced in the FTLD patient-derived fibroblasts from both C9-HRE carriers and non-carriers. Our findings suggest that FTLD patient-derived fibroblasts, regardless of whether they carry the C9-HRE expansion, show unchanged proteasomal and autophagic function, but significantly impaired mitochondrial function and increased accumulation of p62 when compared to control fibroblasts. These findings suggest the possibility of utilizing FTLD patient-derived fibroblasts as a platform for biomarker discovery and testing of drugs targeted to specific cellular functions, such as mitochondrial respiration.

Expression of C9orf72 hexanucleotide repeat expansion leads to formation of RNA foci and dipeptide repeat proteins but does not influence autophagy or proteasomal function in neuronal cells.

C9orf72 hexanucleotide repeat expansion (HRE) is the major genetic cause underpinning frontotemporal lobar degeneration (FLTD) and amyotrophic lateral sclerosis (ALS). C9orf72 HRE-associated pathogenesis involves both loss-of-function, through reduced C9orf72 levels, and gain-of-function mechanisms, including formation of RNA foci and generation of dipeptide repeat (DPR) proteins. In addition, dysfunctional protein degradation pathways, i.e. autophagy and ubiquitin-proteasome system (UPS), are suggested. Our aim was to study the gain-of-function mechanisms in the context of the function of protein degradation pathways as well as the regulation of the DPR proteins through these pathways. To this end, we expressed the pathological HRE in neuronal N2a cells and mouse primary cortical neurons. Protein degradation pathways were modulated to induce or block autophagy or to inhibit UPS. In addition, proteasomal activity was assessed. The C9orf72 HRE-expressing N2a cells and neurons were confirmed to produce RNA foci and DPR proteins, predominantly the Poly-GP proteins. However, the presence of these pathological hallmarks did not result in alterations in autophagy or proteasomal activity in either of the studied cell types. In N2a cells, Poly-GP proteins appeared in soluble forms and Lactacystin-mediated UPS inhibition increased their levels, indicating proteasomal regulation. Similar effects were not observed in cortical neurons, where the Poly-GP proteins formed also higher molecular weight forms. These results suggest a cell type-specific morphology and regulation of the DPR proteins. Further studies in other model systems may shed additional light onto the effects of the C9orf72 HRE on cellular protein degradation pathways and the regulation of the DPR protein levels.

BV-2 Microglial Cells Overexpressing C9orf72 Hexanucleotide Repeat Expansion Produce DPR Proteins and Show Normal Functionality but No RNA Foci.

Hexanucleotide repeat expansion (HRE) in the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause underpinning frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). It leads to the accumulation of toxic RNA foci and various dipeptide repeat (DPR) proteins into cells. These C9orf72 HRE-specific hallmarks are abundant in neurons. So far, the role of microglia, the immune cells of the brain, in C9orf72 HRE-associated FTLD/ALS is unclear. In this study, we overexpressed C9orf72 HRE of a pathological length in the BV-2 microglial cell line and used biochemical methods and fluorescence imaging to investigate its effects on their phenotype, viability, and functionality. We found that BV-2 cells expressing the C9orf72 HRE presented strong expression of specific DPR proteins but no sense RNA foci. Transiently increased levels of cytoplasmic TAR DNA-binding protein 43 (TDP-43), slightly altered levels of p62 and lysosome-associated membrane protein (LAMP) 2A, and reduced levels of polyubiquitinylated proteins, but no signs of cell death were detected in HRE overexpressing cells. Overexpression of the C9orf72 HRE did not affect BV-2 cell phagocytic activity or response to an inflammatory stimulus, nor did it shift their RNA profile toward disease-associated microglia. These findings suggest that DPR proteins do not affect microglial cell viability or functionality in BV-2 cells. However, additional studies in other models are required to further elucidate the role of C9orf72 HRE in microglia.

Frontotemporal dementia: a conference report from the 2nd FinFTD Symposium.

The 2nd FinFTD Symposium was held on 13 September 2019, in Kuopio, Finland, and attracted 80 attendees from six different countries. The program, spanning from molecular mechanisms to biomarkers, prevention, diagnosis and treatment of frontotemporal lobar degeneration and related diseases, provided a great opportunity for researchers, clinicians, healthcare professionals and other participants to discuss about the current status and future directions of frontotemporal lobar degeneration research.

Influence of retinal NMDA receptor activity during autoimmune optic neuritis.

Autoimmune optic neuritis (AON), a model of multiple sclerosis-associated optic neuritis, is accompanied by degeneration of retinal ganglion cells (RGCs) and optic nerve demyelination and axonal loss. In order to investigate the role of N-methyl-d-aspartate (NMDA) receptors in mediating RGC degeneration, upstream changes in the optic nerve actin cytoskeleton and associated deterioration in visual function, we induced AON in Brown Norway rats by immunization with myelin oligodendrocyte glycoprotein. Subsequently, visual acuity was assessed by recording visual evoked potentials and electroretinograms prior to extraction of optic nerves for western blot analysis and retinas for quantification of RGCs. As previously reported, in Brown Norway rats RGC degeneration is observed prior to onset of immune cell infiltration and demyelination of the optic nerves. However, within the optic nerve, destabilization of the actin cytoskeleton could be seen as indicated by an increase in the globular to filamentous actin ratio. Interestingly, these changes could be mimicked by intravitreal injection of glutamate, and similarly blocked by application of the NMDA receptor blocker MK-801, leading us to propose that prior to optic nerve lesion formation, NMDA receptor activation within the retina leads to retinal calcium accumulation, actin destabilization within the optic nerve as well as a deterioration of visual acuity during AON.

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration.

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

New cGMP analogues restrain proliferation and migration of melanoma cells.

Melanoma is one of the most aggressive cancers and displays high resistance to conventional chemotherapy underlining the need for new therapeutic strategies. The cGMP/PKG signaling pathway was detected in melanoma cells and shown to reduce migration, proliferation and to increase apoptosis in different cancer types. In this study, we evaluated the effects on cell viability, cell death, proliferation and migration of novel dimeric cGMP analogues in two melanoma cell lines (MNT1 and SkMel28). These new dimeric cGMP analogues, by activating PKG with limited effects on PKA, significantly reduced proliferation, migration and increased cell death. No decrease in cell viability was observed in non-tumor cells suggesting a tumor-specific effect. These effects observed in melanoma are possibly mediated by PKG2 activation based on the decreased toxic effects in tumor cell lines not expressing PKG2. Finally, PKG-associated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP), linked to cell death, proliferation and migration was found increased and with a change of subcellular localization. Increased phosphorylation of RhoA induced by activation of PKG may also contribute to reduced migration ability of the SkMel28 melanoma cell line when treated with cGMP analogues. These findings suggest that the cGMP/PKG pathway can be envisaged as a therapeutic target of novel dimeric cGMP analogues for the treatment of melanoma.

New dimeric cGMP analogues reduce proliferation in three colon cancer cell lines.

Activation of the cGMP-dependent protein kinase G (PKG) can inhibit growth and/or induce apoptosis in colon cancer. In this study we evaluated the effects on cell viability, cell death and proliferation of novel dimeric cGMP analogues, compared to a monomeric compound. Three colon cancer cell lines, which only express isoform 2 of PKG, were treated with these novel cGMP analogues and responded with increased PKG activity. cGMP analogues reduced cell viability in the three cell lines and this was due to a cytostatic rather than cytotoxic effect. These findings suggest that activation of PKG2 can be a therapeutic target in the treatment of colon cancer and, most importantly, that dimeric cGMP analogues can further improve the beneficial effects previously observed with monomeric cGMP analogues.

Calcium influx and calpain activation mediate preclinical retinal neurodegeneration in autoimmune optic neuritis.

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Recently, the neurodegenerative component of multiple sclerosis has come under focus particularly because permanent disability in patients correlates well with neurodegeneration; and observations in both humans and multiple sclerosis animal models highlight neurodegeneration of retinal ganglion cells as an early event. After myelin oligodendrocyte glycoprotein immunization of Brown Norway rats, significant retinal ganglion cell loss precedes the onset of pathologically defined autoimmune optic neuritis. To study the role calcium and calpain activation may play in mediating early degeneration, manganese-enhanced magnetic resonance imaging was used to monitor preclinical calcium elevations in the retina and optic nerve of myelin oligodendrocyte glycoprotein-immunized Brown Norway rats. Calcium elevation correlated with an increase in calpain activation during the induction phase of optic neuritis, as revealed by increased calpain-specific cleavage of spectrin. The relevance of early calpain activation to neurodegeneration during disease induction was addressed by performing treatment studies with the calpain inhibitor calpeptin. Treatment not only reduced calpain activity but also protected retinal ganglion cells from preclinical degeneration. These data indicate that elevation of retinal calcium levels and calpain activation are early events in autoimmune optic neuritis, providing a potential therapeutic target for neuroprotection.

Preclinical retinal neurodegeneration in a model of multiple sclerosis.

Neurodegeneration plays a major role in multiple sclerosis (MS), in which it is thought to be the main determinant of permanent disability. However, the relationship between the immune response and the onset of neurodegeneration is still a matter of debate. Moreover, recent findings in MS patients raised the question of whether primary neurodegenerative changes can occur in the retina independent of optic nerve inflammation. Using a rat model of MS that frequently leads to optic neuritis, we have investigated the interconnection between neurodegenerative and inflammatory changes in the retina and the optic nerves with special focus on preclinical disease stages. We report that, before manifestation of optic neuritis, characterized by inflammatory infiltration and demyelination of the optic nerve, degeneration of retinal ganglion cell bodies had already begun and ultrastructural signs of axon degeneration could be detected. In addition, we observed an early activation of resident microglia in the retina. In the optic nerve, the highest density of activated microglia was found within the optic nerve head. In parallel, localized breakdown in the integrity of the blood-retinal barrier and aberrations in the organization of the blood-brain barrier marker aquaporin-4 in the optic nerves were observed during the preclinical phase, before onset of optic neuritis. From these findings, we conclude that early and subtle inflammatory changes in the retina and/or the optic nerve head reminiscent of those suggested for preclinical MS lesions may initiate the process of neurodegeneration in the retina before major histopathological signs of MS become manifest.