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TPS5618 Background: Despite radical primary surgery and carboplatin/paclitaxel-based chemotherapy in combination with anti-angiogenic bevacizumab and/or PARP inhibitors (PARPi), most patients (pts) with advanced ovarian cancer (OC) will relapse. Following the implementation of these targeted therapies to first-line treatment, repeated use of bevacizumab and/or PARPi is often not approved nor has conclusively been proven efficacious for all pts with recurrent OC. New combination partners for platinum-based chemotherapy remain important to improve outcome. The antibody-drug conjugate Mirvetuximab soravtansine (MIRV) is comprised of a folate receptor alpha (FRα)-binding antibody, cleavable linker, and the maytansinoid DM4, a potent tubulin-targeting agent. Results from the phase III trial FORWARD I revealed in an exploratory analysis that pts with high FRα expression following PS2+ Scoring (cut off: ≥75% of tumor cells with FRα membrane staining and ≥ 2+ intensity) had significant progression-free survival (PFS) improvements with a hazard ratio of 0.55 compared to mono-chemotherapy (median PFS 5.6 vs 3.2 months, P=0.015) and high activity was most recently confirmed in the SORAYA trial with an overall response rate (ORR) of 32.4%. Preliminary data for the combination of MIRV with carboplatin exist from the Phase 1b FORWARD II trial, which resulted in an ORR of 71%, observed in 17 pts with a median PFS of 15 months, and an ORR of 80% in the FRα medium/high (>50% PS2+) subset of 10 pts. MIRV is well-tolerated with a manageable safety profile. Methods: Eligible pts for this multicenter, randomized, two-arm, open-label, comparative phase II trial must have recurrent, FRα high epithelial cancer of the ovary, fallopian tube or peritoneum and have measurable disease. Pts are eligible for platinum-based chemotherapy with a platinum-free interval of more than 3 months and had at least one prior chemotherapy, but are not candidates to receive bevacizumab for the current relapse. Pts can be included irrespective of wildtype BRCA1/2 mutation status, pts with a deleterious mutation are required to have received prior PARPi therapy. Following pre-screening for high FRα expression in FFPE tumor tissue according to PS2+ scoring, 136 pts are randomized (1:1) to: a) Control arm: Platinum-based combination chemotherapy (for 6 cycles) followed by PARPi if indicated or standard of care or b) Experimental arm: Carboplatin + MIRV 6 mg/kg adjusted ideal body weight (AIBW) IV d1 (6 cycles q21d) followed by MIRV monotherapy 6 mg/kg AIBW IV q21d until disease progression. The primary endpoint of PFS will be assessed by modified RECIST 1.1. Key secondary endpoints include overall survival, ORR, and quality of life. Enrollment started in September 2021. Clinical trial information: NCT04274426.

The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. Certain aspects of cell proliferation in the adult hippocampus are regulated by melatonin and accompanied by apoptosis to ensure proper tissue maintenance and function. The present study investigated Zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis in the adult murine hippocampus and their regulation by melatonin receptor type1 and type2 (MT1/2)-mediated signaling. Adult melatonin-proficient C3H/HeN mice and melatonin-proficient (C3H/HeN) mice with targeted deletion of MT1/2 were adapted to a 12-hours light, 12-hours dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase-3 in combination with different markers for the diverse cell types residing in the hippocampus served to identify and quantify proliferating and apoptotic cells in the hippocampal subregions. ZT-dependent changes in cell proliferation and apoptosis were found exclusively in the subgranular zone (SGZ) and granule cell layer (GCL) of melatonin-proficient mice with functional MT1/2. Cell proliferation in the SGZ showed ZT-dependent changes indicated by an increase of proliferating immature neurons during the dark phase of the 24-hours light-dark cycle. Apoptosis showed ZT-dependent changes in the SGZ and GCL indicated by an increase of apoptotic immature neurons at ZT06 (SGZ) and a decrease of immature and mature neurons at ZT18 (GCL). Our results indicate that ZT-dependent changes in proliferation of immature neurons in the SGZ are counterbalanced by ZT-dependent changes in apoptosis of immature and mature neurons in the SGZ and GCL exclusively in mice with functional MT1/2. Therefore, MT1/2-mediated signaling appears to be crucial for generation and timing of ZT-dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the SGZ. This article is protected by copyright. All rights reserved.

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308.

The polyglutamine (polyQ) diseases are a group of genetically and clinically heterogeneous neurodegenerative diseases, characterized by the expansion of polyQ sequences in unrelated disease proteins, which form different types of neuronal aggregates. The aim of this study was to characterize the aggregation pathology in the brainstem of spinocerebellar ataxia type 2 (SCA2) and 3 (SCA3) patients. For good recognition of neurodegeneration and rare aggregates, we employed 100 µm PEG embedded brainstem sections, which were immunostained with the 1C2 antibody, targeted at polyQ expansions, or with an antibody against p62, a reliable marker of protein aggregates. Brainstem areas were scored semiquantitatively for neurodegeneration, severity of granular cytoplasmic staining (GCS) and frequency of neuronal nuclear inclusions (NNI). SCA2 and SCA3 tissue exhibited the same aggregate types and similar staining patterns. Several brainstem areas showed statistically significant differences between disease groups, whereby SCA2 showed more severe GCS and SCA3 showed more numerous NNI. We observed a positive correlation between GCS severity and neurodegeneration in SCA2 and SCA3 and an inverse correlation between the frequency of NNI and neurodegeneration in SCA3. Although their respective disease proteins are unrelated, SCA2 and SCA3 showed the same aggregate types. Apparently, the polyQ sequence alone is sufficient as a driver of protein aggregation. This is then modified by protein context and intrinsic properties of neuronal populations. The severity of GCS was the best predictor of neurodegeneration in both disorders, while the inverse correlation of neurodegeneration and NNI in SCA3 tissue implies a protective role of these aggregates.

Background/Aims: Zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis are regulated by melatonin receptor (MT)-mediated signaling in the adult hippocampus and hypothalamic-hypophyseal system. There are two G-protein-coupled MT subtypes, MT1 and MT2. Therefore, the present study examined which MT subtype is required for the regulation of ZT-dependent changes in cell proliferation and/or apoptosis in the adult murine brain and pituitary. Methods: Adult melatonin-proficient (C3H) mice with targeted deletion of MT1 (MT1 KO) or MT2 (MT2 KO) were adapted to a 12-h light/12-h dark photoperiod and sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 or activated caspase-3 served to quantify proliferating and apoptotic cells in the hippocampal subgranular zone (SGZ) and granule cell layer, the hypothalamic median eminence (ME), and the hypophyseal pars tuberalis. Results: ZT-dependent changes in cell proliferation were found exclusively in the SGZ and ME of MT1 KO mice, while apoptosis showed no ZT-dependent changes in the regions analyzed, neither in MT1 nor in MT2 KO mice. Comparison with our previous studies in C3H mice with functional MTs and MT1/2 KO mice revealed that MT2-mediated signaling is required and sufficient for ZT-dependent changes in cell proliferation in the SGZ and ME, while ZT-dependent changes in apoptosis require signaling from both MT subtypes. Conclusions: Our results indicate that generation and timing of ZT-dependent changes in cell proliferation and apoptosis by melatonin require different MT subtype constellations and emphasize the importance to shed light on the specific function of each receptor subtype in different tissues and physiological conditions.

AimsPolyglutamine (polyQ) diseases are characterized by the expansion of a polymorphic glutamine sequence in disease-specific proteins and exhibit aggregation of these proteins. This is combated by the cellular protein quality control (PQC) system, consisting of chaperone-mediated refolding as well as proteasomal and lysosomal degradation pathways. Our recent study in the polyQ disease spinocerebellar ataxia type 3 (SCA3) suggested a distinct pattern of protein aggregation and PQC dysregulation. MethodsTo corroborate these findings we have investigated immunohistochemically stained 5m sections from different brain areas of Huntington's disease (HD) and SCA3 patients. ResultsIrrespective of disease and brain region, we observed peri- and intranuclear polyQ protein aggregates. A subset of neurones with intranuclear inclusions bodies exhibited signs of proteasomal dysfunction, up-regulation of HSPA1A and re-distribution of DNAJB1. The extent of the observed effects varied depending on brain area and disease protein. ConclusionsOur results suggest a common sequence, in which formation of cytoplasmic and nuclear inclusions precede proteasomal impairment and induction of the cellular stress response. Clearly, impairment of the PQC is not the primary cause for inclusion formation, but rather a consequence that might contribute to neuronal dysfunction and death. Notably, the inclusion pathology is not directly correlated to the severity of the degeneration in different areas, implying that different populations of neurones respond to polyQ aggregation with varying efficacy and that protein aggregation outside the neuronal perikaryon (e.g. axonal aggregates) or other effects of polyQ aggregation, which are more difficult to visualize, may contribute to neurodegeneration.

Background/Aims: Cell proliferation and apoptosis are known to adjust neuroendocrine circuits to the photoperiod. The latter is communicated by melatonin, the hormone secreted by the pineal organ. The present study investigated zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis in the adult murine neuroendocrine system and their regulation by melatonin. Methods: Adult melatonin-proficient (C3H/HeN) and melatonin-deficient (C57Bl/6J) mice, as well as melatonin-proficient (C3H/HeN) mice with targeted deletion of both melatonin receptor types (MT1 and MT2) were adapted to a 12-hour light, 12-hour dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase-3 served to identify and quantify proliferating and apoptotic cells in the median eminence (ME), hypophyseal pars tuberalis, and pars distalis (PD). Results: ZT-dependent changes in cell proliferation and apoptosis were found exclusively in melatonin-proficient mice with functional MTs. Cell proliferation in the ME and PD showed ZT-dependent changes indicated by an increase at ZT12 (ME) and a decrease at ZT06 (PD). Apoptosis showed ZT-dependent changes in all regions analyzed, indicated by an increase at ZT06. Proliferating and apoptotic cells were found in nearly all cell types residing in the regions analyzed. Conclusions: Our results indicate that ZT-dependent changes in cell proliferation are counterbalanced by ZT-dependent changes in apoptosis exclusively in melatonin-proficient mice with functional MTs. Melatonin signaling appears to be crucial in both the generation and timing of proliferation and apoptosis that serve the high rate of physiological cell turnover in the adult neuroendocrine system.

Ablating the cochlea causes total sensory deafferentation of the cochlear nucleus. Over the first postoperative week, degeneration of the auditory nerve and its synaptic terminals in the cochlear nucleus temporally overlaps with its re-innervation by axon collaterals of medial olivocochlear neurons. At the same time, astrocytes increase in size and density. We investigated the time courses of the expression of ezrin, polysialic acid, matrix metalloprotease-9 and matrix metalloprotease-2 within these astrocytes during the first week following cochlear ablation. All four proteins are known to participate in degeneration, regeneration, or both, following injury of the central nervous system. In a next step, stereotaxic injections of kainic acid were made into the ventral nucleus of the trapezoid body prior to cochlear ablation to destroy the neurons that re-innervate the deafferented cochlear nucleus by axon collaterals developing growth-associated protein 43 immunoreactivity. This experimental design allowed us to distinguish between molecular processes associated with degeneration and those associated with re-innervation. Under these conditions, astrocytic growth and proliferation showed an unchanged deafferentation-induced pattern. Similarly, the distribution and amount of ezrin and matrix metalloprotease-9 in astrocytes after cochlear ablation developed in the same way as under cochlear ablation alone. In sharp contrast, the astrocytic expression of polysialic acid and matrix metalloprotease-2 normally invoked by cochlear ablation collapsed when re-innervation of the cochlear nucleus was inhibited by lesioning medial olivocochlear neurons with kainic acid. In conclusion, re-innervation, including axonal growth and synaptogenesis, seems to prompt astrocytes to recompose their molecular profile, paving the way for tissue reorganisation after nerve degeneration and loss of synaptic contacts.

The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. Certain aspects of cell proliferation in the adult hippocampus are regulated by melatonin and accompanied by apoptosis to ensure proper tissue maintenance and function. The present study investigated Zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis in the adult murine hippocampus and their regulation by melatonin receptor type1 and type2 (MT1/2)-mediated signaling. Adult melatonin-proficient C3H/HeN mice and melatonin-proficient (C3H/HeN) mice with targeted deletion of MT1/2 were adapted to a 12-h light, 12-h dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase-3 in combination with different markers for the diverse cell types residing in the hippocampus served to identify and quantify proliferating and apoptotic cells in the hippocampal subregions. ZT-dependent changes in cell proliferation and apoptosis were found exclusively in the subgranular zone (SGZ) and granule cell layer (GCL) of melatonin-proficient mice with functional MT1/2. Cell proliferation in the SGZ showed ZT-dependent changes indicated by an increase of proliferating immature neurons during the dark phase of the 24-h light-dark cycle. Apoptosis showed ZT-dependent changes in the SGZ and GCL indicated by an increase of apoptotic immature neurons at ZT06 (SGZ) and a decrease of immature and mature neurons at ZT18 (GCL). Our results indicate that ZT-dependent changes in proliferation of immature neurons in the SGZ are counterbalanced by ZT-dependent changes in apoptosis of immature and mature neurons in the SGZ and GCL exclusively in mice with functional MT1/2. Therefore, MT1/2-mediated signaling appears to be crucial for generation and timing of ZT-dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the SGZ. © 2017 Wiley Periodicals, Inc.

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308.

The polyglutamine (polyQ) diseases are a group of genetically and clinically heterogeneous neurodegenerative diseases, characterized by the expansion of polyQ sequences in unrelated disease proteins, which form different types of neuronal aggregates. The aim of this study was to characterize the aggregation pathology in the brainstem of spinocerebellar ataxia type 2 (SCA2) and 3 (SCA3) patients. For good recognition of neurodegeneration and rare aggregates, we employed 100 µm PEG embedded brainstem sections, which were immunostained with the 1C2 antibody, targeted at polyQ expansions, or with an antibody against p62, a reliable marker of protein aggregates. Brainstem areas were scored semiquantitatively for neurodegeneration, severity of granular cytoplasmic staining (GCS) and frequency of neuronal nuclear inclusions (NNI). SCA2 and SCA3 tissue exhibited the same aggregate types and similar staining patterns. Several brainstem areas showed statistically significant differences between disease groups, whereby SCA2 showed more severe GCS and SCA3 showed more numerous NNI. We observed a positive correlation between GCS severity and neurodegeneration in SCA2 and SCA3 and an inverse correlation between the frequency of NNI and neurodegeneration in SCA3. Although their respective disease proteins are unrelated, SCA2 and SCA3 showed the same aggregate types. Apparently, the polyQ sequence alone is sufficient as a driver of protein aggregation. This is then modified by protein context and intrinsic properties of neuronal populations. The severity of GCS was the best predictor of neurodegeneration in both disorders, while the inverse correlation of neurodegeneration and NNI in SCA3 tissue implies a protective role of these aggregates.

Lesion-induced neuroplasticity, including fiber degeneration, axonal growth, and synaptogenesis, involves dynamical changes of the extracellular matrix. We discovered that the matrix metalloprotease-2 (MMP-2), a major actor in extracellular matrix recomposition, is changed in distribution and increased in amount in the ventral cochlear nucleus after unilateral cochlear ablation. There was a remarkable coincidence of MMP-2 accumulation and GAP-43 expression in time and space. We obtained evidence indicating that MMP-2 is delivered to regions of emerging GAP-43 positive synaptic endings by postsynaptic neurons as well as by adjoining astrocytes. These results indicate a major role of MMP-2 in lesion-induced remodeling of central auditory networks and suggest a cooperativity with GAP-43-directed axonal outgrowth and synaptogenesis.

Objective: It has been debated for decades if and to what extent the mature central nervous system of mammals is capable to respond to altered sensory use and function with changes of structure and function. We attempted to shed new light on the neuroplastic potential of the central auditory system, using light and electron microscopic techniques to explore it on the levels of cells (both neurons and glia), synapses, selected molecular components of the tissue, and the spatiotemporal relationship among these following the induction of changed patterns of sensory activity. Study Design: Working in an animal model, we induced two modifications to normal auditory signalling in the adult rat brain. Through unilateral cochleotomy, we caused a total primary deafferentation in the cochlear nucleus and abolished all sensory input through the ear. By electrical intracochlear stimulation (EIS) we substituted normal sensory signalling by an artificial input that was under complete temporal control. Results: ...

The nuclei of the auditory brainstem harbor a diversity of neuronal cell types and are interconnected by excitatory as well as inhibitory ascending, descending, and commissural pathways. Classically, neurons have been characterized by size and shape of their cell body and by the geometry of their dendrites. Our study is based on the use of axonal tracers in combination with immunocytochemistry to identify and distinguish neuronal subtypes by their molecular signature in dorsal and ventral cochlear nucleus, lateral superior olive, medial superior olive, medial nucleus of the trapezoid body, and inferior colliculus of the adult rat. The presumed neurotransmitters glutamate, glycine, and GABA were used alongside the calcium-binding proteins parvalbumin, calretinin, and calbindin-D28k as molecular markers. Our data provide distinct extensions to previous characterizations of neuronal subtypes and reveal regularities and differences across auditory brainstem nuclei that are discussed for their functional implications.