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  Glial and vascular contributions to neurodegenerative diseases
  western blotting, TEM, d-STORM and Zetaview. Proteomic analysis was completed on EVs and cell lysates from each model using mass spectrometry (LC-MS/MS and SWATH MS).
Serum-cultured astrocytes showed morphological changes and upregulated markers of astrocyte reactivity compared to serum- free culture (n=5). RNA-SEQ found 1474 unique protein-coding RNA exclusive to serum-free cultures (n=4). Gene ontology analysis found 577 significantly upregulated pathways in serum-free astrocytes including cell-adhesion pathways, axon and synapse development and ion channel activity. Proteomic analysis found distinct differences in EV protein cargo and cell lysates in both models with ~250 proteins identified in EV samples (n=3).
These findings suggest that there are clear differences in astrocytes when using serum media and should not be used to study quiescent astrocytes and their ADEVs. Instead, we recommend serum-free cultures whilst serum-based cultures can be used as a more general model of reactive astrocytes.
Pathological roles of microglial gene INPP5D in a mouse model of Alzheimer disease
Sho Takatori1, Akihiro Iguchi1, Shingo Kimura1, Hiroki Muneto1, Kai Wang1, Yukiko Hori1, Junko Sasaki2, Takashi Saito3,4, Takaomi Saido3, Tsuneya Ikezu5, Toshiyuki Takai6, Takehiko Sasaki2, Taisuke Tomita1
1Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Japan, 2Department of Lipid Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Japan, 3Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, 2-1 Hirosawa, Wako, Japan, 4Department of Neurocognitive Science, Institute of Brain Science, Nagoya City University Graduate School of Medical Science, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Japan, 5Department of Neuroscience, Mayo Clinic Florida, Jacksonville, USA, 6Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo, Sendai, Japan
Experimental traumatic brain injury-induced microglia inflammation in the olfactory
bulb disrupts neuronal networks leading to olfactory dysfunction
Junfang Wu1, Xiang Liu1, Dylan Gilhooly2, Junyun He1, Zhuofan Lei1, Yun Li1, Rodney Ritzel1, Long-Jun Wu3, Shaolin Liu2
1University of Maryland School of Medicine, Baltimore, USA, 2Howard University College of Medicine, Washington, USA, 3Mayo Clinic, Rochester, USA
 Background: Approximately 20–68% of traumatic brain injury (TBI) patients exhibit trauma-associated olfactory deficits (OD) which can compromise not only the quality of life but also cognitive and neuropsychiatric functions. Although post-traumatic anosmia has been documented in the medical literature for more than a century, few studies have examined the impact of TBI on OD. The present study examined microglia inflammation and neuronal dysfunction in the olfactory bulb (OB) as well as the underlying mechanisms associated with OD in mice using a controlled cortical impact (CCI) model. Methods: Young adult male mice were subjected to mild/moderate- level CCI. Flow cytometry, qPCR, and IHC were used to examine inflammation in the OB after TBI. Electrophysiological recording was applied to characterize OB neuronal excitability. Transgenic mice with ablation of the voltage-gated proton channel Hv1 or NOX2 as well as a specific NOX2 inhibitor were used to determine the effects of Hv1 and NOX2 activity on OD. Olfactory function was assessed by buried food test, two-bottle odor discrimination test, and odor memory for up to six-months post-injury.
Results: TBI caused a rapid inflammatory response in the OB including elevated proinflammatory cytokines, numbers of microglia and infiltrating myeloid cells. Upregulation of Hv1 and NOX2 expression levels at 1 d post-injury was observed predominantly in microglia/ macrophages. Genetic ablation of Hv1 significantly attenuated the production of ROS and cytokines. TBI-induced early neuronal hyperexcitation and later hypo-neuronal activity in the OB were mitigated in the absence of Hv1. In a battery of olfactory behavioral tests, WT/TBI mice displayed significant OD, which, in contrast, was observed in neither Hv1 KO/TBI nor NOX2 KO/TBI mice. Finally, intranasal delivery of a NOX2 inhibitor ameliorated post-traumatic OD.
Conclusions: Our data indicate that Hv1/NOX2-mediated microglia inflammation in the OB disrupts OB neuronal circuits leading to poorer olfactory function late after TBI.
Introduction: Brain deposition of amyloid β (Aβ) is crucial for the Alzheimer disease (AD) pathogenesis. Recent finding that genetic variants of TREM2, encoding a microglial receptor, increase AD risks suggests a pathological involvement of microglia. Functional deficiencies of TREM2 disrupt microglial envelopment of Aβ plaques, impair their transcriptional response to Aβ, and worsen neuritic dystrophy, implying that TREM2 is essential for neuroprotection by microglia. However, the underlying molecular mechanism remains unclear.
Methods: We investigated pathological roles of another AD risk gene, INPP5D, a negative regulator of phosphoinositide PI(3,4,5) P3 and a downstream effector of TREM2 signaling. Effects of Inpp5d heterodeficiency were examined in an AD mouse model (App NLGF, hereafter NLGF mice), as well as by crossing with TREM2 loss-of-function model (Tyrobp-deficient NLGF mice) to ask their genetic interaction in in vivo.
Results and Discussion: In Tyrobp-deficient NLGF mice, Inpp5d haplodeficiency restored microglial clustering around Aβ plaques, partially restored several abnormalities previously documented in Trem2-deficient mice (plaque shapes and astrogliosis), and reduced deposition of phosphorylated tau, another pathological hallmark of AD. Mechanistic analyses suggest that TREM2/TYROBP and INPP5D regulate microglial association with Aβ plaques by oppositely regulating PI(3,4,5) P3 signaling as well as microglial adhesion to extracellular materials. Conclusion: Our results suggest that INPP5D acts downstream of TREM2/TYROBP to regulate the microglial barrier against Aβ toxicity, thereby affects Aβ-dependent deposition of tau.
Astrocytic changes in mitochondrial oxidative phosphorylation and quality control proteins in Parkinson's disease
Chun Chen1, David McDonald2, Alasdair Blain1, Andrew Filby2, Amy Vincent1, Gavin Hudson1, Amy Reeve1
1Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, United Kingdom, 2Flow Cytometry Core Facility, Newcastle University, Newcastle upon Tyne, United Kingdom
 66 • ISMND 2022
Background: Mitochondrial dysfunction within neurons, particularly those of the substantia nigra, has been well characterized in Parkinson's disease (PD) and is considered to be related to the pathogenesis of this disorder. Dysfunction within this important organelle impairs neuronal communication and survival, however, the reliance of astrocytes on mitochondria and the impact of their dysfunction on this cell type are less well characterized. This study aimed to uncover whether astrocytes harbour oxidative phosphorylation (OXPHOS) deficiencies and show

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