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  Glial and vascular contributions to neurodegenerative diseases
The Trk-PAM ACD856 improves mitochondrial function and increase BDNF levels in primary cortical neurons
Cristina Parrado1, Sanja Juric1, Märta Dahlström1, Johan Sandin1,2, Pontus Forsell1,2
1Alzecure Pharma AB, Huddinge, Sweden, 2Karolinska Institutet, Dept of Neurobiology, Care sciences and society, div. of Neurogeriatrics, Solna, Sweden
 Objectives: Although brain-derived neurotrophic factor (BDNF) [1] and nerve growth factor (NGF) [2] were originally characterized based upon their effects on neuronal cells, they are now also described to have non-neuronal effects. BDNF and NGF have been shown to play a role in conditions such as Alzheimer’s disease [3], inflammatory disorders [4] [5] and depression[6], as well as controlling bioenergetics [7]. ACD856 is a positive allosteric modulator (PAM) of the receptors for NGF and BDNF, i.e. TrkA and TrkB, respectively. Binding of ACD856 led to increased phosphorylation of the receptors, increased release of neurotransmitters, induction of long-term potentiation and improved memory [8]. ACD856 was demonstrated to be well tolerated and to have favorable pharmacokinetic profile in a clinical phase 1 study.
Aim: The aim was to study effects of ACD856 on mitochondrial function and BDNF levels in cortical neurons.
Methods: Effects of BDNF or ACD856 on mitochondrial function was investigated in cortical neurons with glutamine as the main energy source. ATP levels and cellular permeability was used to measure effects on bioenergetics. Effects on BDNF levels were studied by incubating cortical neurons with ACD856 and analyzing BDNF by ELISA.
Results: ACD856 demonstrated a dose-dependent positive effect on mitochondrial function as measured by an increase in ATP levels and a decrease in cellular permeability, suggesting a general beneficial effect on neuronal health. Furthermore, ACD856 could increase the levels of BDNF in a dose-dependent manner.
Conclusion: ACD856 is a positive modulator of Trk-receptors with beneficial effects on neuronal health. Surprisingly, the compound also increased the levels of BDNF in cortical neurons, suggesting a feed-forward effect of the compound on BDNF-TrkB signaling. Our findings support that ACD856 may provide a neuroprotective effect by improving mitochondrial function and increasing levels of BDNF. Our findings suggest that ACD856 could have disease-modifying effects besides the pro-cognitive effects.
Materials and Methods: We crossed a novel Cre-dependent genetic sparse labeling MORF3 mice (Veldman et al., 2020, PMID: 32795398) to mouse models of Huntington’s disease (mutant Huntingtin Q140 knockin) and Alzheimer’s disease (5xFAD) to sparsely and brightly label the striatal medium spiny neurons (MSNs) and layer 5 cortical pyramidal neurons, respectively.
Results: We applied automated tissue clearing and immunostaining followed by light-sheet imaging of brain hemispheres or thick brain sections to obtain datasets for MORF3-labeled neurons. We also implemented a novel semi-automated digital reconstruction and morphological analysis pipeline. We then applied the pipeline to analyze hundreds of striatal MSNs in MORF3/Camk2a-CreER+ brains in either Q140 or wildtype backgrounds. The reconstructed MSNs are mapped to anatomically defined striatal subregions. Our study revealed multiple MSN dendritic pathologies (e.g. dendric path length and local dendritic bifurcation angles) that are selective to specific striatal subregions and are accompanied by dendritic spine loss. Our ongoing analyses include imaging and morphological analysis of MORF-labeled cortical layer 5 neurons (MORF3/Etv1-CreER) and microglia (MORF3/ Cx3cr1-CreER) in wildtype and 5xFAD backgrounds.
Conclusions: Our study provides a novel systems biological approach to study brainwide single-neuron morphologies in wildtype and diseased brains, and provides proof-of-concept on the importance of the large-scale and anatomically-delineated analyses of single-neuron morphological defects in HD mice.
Background: Diabetic retinopathy (DR) is a leading cause of blindness in the working age population. During DR, retinal neurons and the vasculature degenerate and it is recognized that the progressive loss of the homeostatic role of the Müller cells plays a key role in this pathology. Interleukin-33 (IL-33), an immunomodulatory cytokine, is expressed predominately by Müller cells and plays a vital role in retinal homeostasis. This study aimed to understand the role of IL-33 in Müller cell in DR.
Materials and Methods: Diabetes was induced in wild-type (WT) and IL-33-/- mice with intraperitoneal injection of streptozotocin (STZ). Visual function was evaluated by electroretinography. Retinal neuronal and vascular degeneration was examined by immunohistochemistry. Müller cell activation was evaluated using qRT-PCR, immunocyto/ histochemistry and ELISA. Single cell RNAseq datasets from control and diabetic mice were used to analyze Müller cell activation and IL-33 expression.
Results: WT and IL-33-/- mice developed equivalent levels of diabetes following STZ injection. IL-33 expression was increased in Müller cells of diabetic retina using scRNAseq, immunohistochemistry, and qRT-qPCR. ERG a- and b-wave amplitudes, retinal neuronal thickness, and the number of photoreceptors and ganglion cells were significantly lower in IL-33-/- diabetic mice compared to WT diabetic counterparts. Diabetes- induced retinal vascular degeneration was comparable between WT and IL-33-/- mice. The IL-33-/- diabetic retina expressed significantly higher levels of Ccl2, Il1b, il6, Tnfα, GFAP and F4/80. Müller cells from IL- 33-/- mice expressed significantly lower levels of glutamine synthetase (GS) and glutamate transporters (GLAST), as well as neurotrophic growth factors Bdnf, Cntf, and Ngf under normal and high glucose conditions.
Discussion/conclusions: Our results suggest that IL-33 is critical for Müller cell glutamate metabolism and neurotrophic growth factor production. Diabetes upregulates IL-33 expression in Müller cells to maintain retinal homeostasis. Deletion of IL-33 accelerates Müller cell malfunction and retinal neuronal degeneration in diabetes.
IL-33 deficiency accelerates diabetes-induced retinal neuronal degeneration
Mei Chen1, Josy Augustine1, Sofia Pavlou1, Kevin Harkin1,
Alan Stitt1, Heping Xu1
1Queens University Belfast, UK
Brainwide Genetic Sparse Labeling, Imaging and Quantitative Analyses of Single-Neuron Morphological Deficits in Mouse Models of Neurodegenerative Diseases
Chang Park1, Daniel Lee1, Chris Choi1, Peter Langfelder1, Amberlene De La Rocha1, Ming Yan1, X. William Yang1,2 1Center for Neurobehavioral Genetics, Jane and Terry Semel Institute of Neuroscience and Human Behavior; Dept. Psychiatry and Biobehavioral Science, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, USA, 2Brain Research Institute, University of California, Los Angeles, Los Angeles, USA
 Background: The atrophy and loss of dendritic and axonal processes in selective vulnerable neuronal populations is an early and common pathological manifestation of neurodegenerative, neurodevelopmental and neuropsychiatric disorders. It also serves as an important preclinical and clinical readout for developing therapeutics. Despite the importance of single-neuron morphologies in understanding the mammalian brain and brain diseases, there is currently a lack of a generalizable systems-biology approach to analyze brainwide single- neuron morphologies in genetically-defined mammalian neurons at large-scale, from labeling and imaging to extraction and quantitative analyses of morphological statistics.

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