BACKGROUND:Alzheimer's Disease (AD) is a neurodegenerative disease with drastically altered astrocytic metabolism. Astrocytic GABA and H2O2 are associated with memory impairment in AD and synthesized through the Monoamine Oxidase B (MAOB)-mediated multi-step degradation of putrescine. However, the enzymes downstream to MAOB in this pathway remain unidentified. METHODS:Using transcriptomics analysis, we identified two candidate enzymes, Aldehyde Dehydrogenase 1 family member A1 (ALDH1A1) and Sirtuin 2 (SIRT2) for the steps following MAOB in the astrocytic GABA production pathway. We used immunostaining, metabolite analysis and electrophysiology, both in vitro and in vivo, to confirm the participation of these enzymes in astrocytic GABA production. We checked for the presence of SIRT2 in human AD patients as well as the mouse model APP/PS1 and finally, we selectively ablated SIRT2 in the astrocytes of APP/PS1 mice to observe its effects on pathology. RESULTS:Immunostaining, metabolite analysis, and electrophysiology recapitulated the participation of ALDH1A1 and SIRT2 in GABA production. Inhibition of SIRT2 reduced the production of astrocytic GABA but not H2O2, a key molecule in neurodegeneration. Elevated expression of these enzymes was found in hippocampal astrocytes of AD patients and APP/PS1 mice. Astrocyte-specific gene-silencing of SIRT2 in APP/PS1 mice restored GABA production and partially improved memory function. CONCLUSIONS:Our study is the first to identify the specific role of SIRT2 in reactive astrogliosis and determine the specific pathway and metabolic step catalyzed by the enzyme. We determine the partial, yet significant role of ALDH1A1 in this process, thereby highlighting 2 new players the astrocytic GABA production pathway. Our findings therefore, offer SIRT2 as a new tool to segregate GABA from H2O2 production, aiding future research in neurodegenerative diseases.

Impaired ion channels regulating Golgi pH lead to structural alterations in the Golgi apparatus, such as fragmentation, which is found, along with cognitive impairment, in Alzheimer's disease. However, the causal relationship between altered Golgi structure and cognitive impairment remains elusive due to the lack of understanding of ion channels in the Golgi apparatus of brain cells. Here, we identify that a transmembrane protein TMEM87A, renamed Golgi-pH-regulating cation channel (GolpHCat), expressed in astrocytes and neurons that contributes to hippocampus-dependent memory. We find that GolpHCat displays unique voltage-dependent currents, which is potently inhibited by gluconate. Additionally, we gain structural insights into the ion conduction through GolpHCat at the molecular level by determining three high-resolution cryogenic-electron microscopy structures of human GolpHCat. GolpHCat-knockout mice show fragmented Golgi morphology and altered protein glycosylation and functions in the hippocampus, leading to impaired spatial memory. These findings suggest a molecular target for Golgi-related diseases and cognitive impairment.

Alzheimer’s disease (AD) is characterized by the loss of memory due to aggregation of misphosphorylated tau and amyloid beta (Aβ) plaques in the brain, elevated release of inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and reactive oxygen species from astrocytes, and subsequent neurodegeneration. Recently, it was found that enzyme Ornithine Decarboxylase 1 (ODC1) acts as a bridge between the astrocytic urea cycle and the putrescine-to-GABA conversion pathway in the brain of AD mouse models as well as human patients. In this study, we show that the long-term knockdown of astrocytic Odc1 in APP/PS1 animals was sufficient to completely clear Aβ plaques in the hippocampus while simultaneously switching the astrocytes from a detrimental reactive state to a regenerative active state, characterized by proBDNF expression. Our experiments also reveal an effect of astrocytic ODC1 inhibition on the expression of genes involved in synapse pruning and organization, histone modification, apoptotic signaling and protein processing. These genes are previously known to be associated with astrocytic activation and together create a neuroregeneration-supportive environment in the brain. By inhibiting ODC1 for a long period of 3 months in AD mice, we demonstrate that the beneficial amyloid-clearing process of astrocytes can be completely segregated from the systemically harmful astrocytic response to insult. Our study reports an almost complete clearance of Aβ plaques by controlling an endogenous degradation process, which also modifies the astrocytic state to create a regeneration-supportive environment in the brain. These findings present the potential of modulating astrocytic clearance of Aβ as a powerful therapeutic strategy against AD.

Astrocytes have been known to support neuronal function, but until now, memory storage and recall has thought to be largely controlled by neurons. In this article, we shed light on recent research published by Williamson et al. that, for the first time, shows astrocytes to participate in memory formation and recall.

BACKGROUND:Astrocytes, one of the most resilient cells in the brain, transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in Alzheimer's disease (AD). However, reactive astrocyte-mediated non-cell autonomous neuropathological mechanism is not fully understood yet. We aimed our study to find out whether Aβ-induced proteotoxic stress affects the expression of autophagy genes and the modulation of autophagic flux in astrocytes, and if yes, how Aβ-induced autophagy-associated genes are involved Aβ clearance in astrocytes of animal model of AD. METHODS:Whole RNA sequencing (RNA-seq) was performed to detect gene expression patterns in Aβ-treated human astrocytes in a time-dependent manner. To verify the role of astrocytic autophagy in an AD mouse model, we developed AAVs expressing shRNAs for MAP1LC3B/LC3B (LC3B) and Sequestosome1 (SQSTM1) based on AAV-R-CREon vector, which is a Cre recombinase-dependent gene-silencing system. Also, the effect of astrocyte-specific overexpression of LC3B on the neuropathology in AD (APP/PS1) mice was determined. Neuropathological alterations of AD mice with astrocytic autophagy dysfunction were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through novel object recognition test (NOR) and novel object place recognition test (NOPR). RESULTS:Here, we show that astrocytes, unlike neurons, undergo plastic changes in autophagic processes to remove Aβ. Aβ transiently induces expression of LC3B gene and turns on a prolonged transcription of SQSTM1 gene. The Aβ-induced astrocytic autophagy accelerates urea cycle and putrescine degradation pathway. Pharmacological inhibition of autophagy exacerbates mitochondrial dysfunction and oxidative stress in astrocytes. Astrocyte-specific knockdown of LC3B and SQSTM1 significantly increases Aβ plaque formation and GFAP-positive astrocytes in APP/PS1 mice, along with a significant reduction of neuronal marker and cognitive function. In contrast, astrocyte-specific overexpression of LC3B reduced Aβ aggregates in the brain of APP/PS1 mice. An increase of LC3B and SQSTM1 protein is found in astrocytes of the hippocampus in AD patients. CONCLUSIONS:Taken together, our data indicates that Aβ-induced astrocytic autophagic plasticity is an important cellular event to modulate Aβ clearance and maintain cognitive function in AD mice.

Summary paragraph Hemoglobin (Hb) is well-known for transporting oxygen in red blood cells within blood vessels 1 . Although Hb is also present in the brain 2 , its role remains poorly understood. Here, we show that Hb, found in astrocytes of neurodegenerative animal models and patients, displays significant antioxidant effects through its H 2 O 2 -decomposing peroxidase activity, and a small molecule enhancer boosts this activity, reducing aberrant H 2 O 2 and mitigating H 2 O 2 -induced neurodegeneration. To counteract the harmful effects of aberrant H 2 O 2 -production in Alzheimer’s disease (AD), we developed KDS12025, a blood-brain barrier (BBB)-permeable small molecule that effectively enhances the peroxidase activity of Hb by a hundredfold, especially at a low level of Hb. KDS12025 and its analogs achieve this enhancement through its electron-donating amine group. KDS12025 reduces H 2 O 2 levels in astrocytes, exhibits neuroprotective effects, and reverses memory impairment in AD models. Gene-silencing of Hbβ abrogates KDS12025’s impact in both culture and animal models of AD. Moreover, KDS12025 prevented the death of dopaminergic neurons in a Parkinson’s disease (PD) model without altering the oxygen-transporting function of Hb. KDS12025 extended survival and improved motor function even in the severe amyotrophic lateral sclerosis (ALS) mouse model. Our findings propose Hb as a new therapeutic target for neurodegenerative diseases, with KDS12025 emerging as a first-in-class drug candidate that enhances Hb’s peroxidase activity to reduce H 2 O 2 . Boosting Hb’s peroxidase activity with KDS12025 mitigates oxidative stress and alleviates neurodegeneration in AD, PD, and ALS with broad applicability for numerous oxidative-stress-driven diseases.

The Golgi apparatus is a critical intracellular organelle that is responsible for modifying, packaging, and transporting proteins to their destinations. Golgi homeostasis involving the acidic pH, ion concentration, and membrane potential, is critical for proper functions and morphology of the Golgi. Although transporters and anion channels that contribute to Golgi homeostasis have been identified, the molecular identity of cation channels remains unknown. Here we identify TMEM87A as a novel Golgi-resident cation channel that contributes to pH homeostasis and rename it as GolpHCat ( Gol gi pH -sensitive Cat ion channel). The genetic ablation of GolpHCat exhibits an impaired resting pH in the Golgi. Heterologously expressed GolpHCat displays voltage- and pH-dependent, non-selective cationic, and inwardly rectifying currents, with potent inhibition by gluconate. Furthermore, reconstitution of purified GolpHCat in liposomes generates functional channel activities with unique voltage-dependent gating and ion permeation. GolpHCat is expressed in various cell types such as neurons and astrocytes in the brain. In the hippocampus, GolpHCat-knockout mice show dilated Golgi morphology and altered glycosylation and protein trafficking, leading to impaired spatial memory with significantly reduced long-term potentiation. We elucidate that GolpHCat, by maintaining Golgi membrane potential, regulates ionic and osmotic homeostasis, protein glycosylation/trafficking, and brain functions. Our results propose a new molecular target for Golgi-related diseases and cognitive impairment.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-β is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.

ABSTRACT GABA (γ-aminobutyric acid) is the primary inhibitory neurotransmitter in the CNS. In astrocytes, GABA is synthesized by degradation of putrescine by monoamine oxidase B (MAO-B), a process which is known to mediate tonic inhibition of neuronal excitability. This astrocytic tonic GABA and related enzymes are also reported to be involved in memory impairment in Alzheimer’s Disease, and therefore are potential therapeutic targets to rescue memory in AD patients. However, the enzymes downstream of MAO-B in this pathway have not been elucidated yet. To fill this gap in knowledge, we performed transcriptomic and literature database analysis and identified Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) and a histone deacetylase enzyme Sirtuin2 (SIRT2) as plausible candidate enzymes in primary cultured astrocytes. Immunostaining, metabolite analyses, and sniffer patch clamp performed in the presence or absence of suitable inhibitors, or with genetic ablation of the candidate enzymes recapitulated their participation in GABA production. We propose ALDH1A1 and SIRT2 as potential therapeutic targets against Alzheimer’s Disease.

The lateral hypothalamic area (LHA) regulates food intake and energy balance. Although LHA neurons innervate adipose tissues, the identity of neurons that regulate fat is undefined. Here we show that GABRA5-positive neurons in LHA (GABRA5 LHA ) polysynaptically project to brown and white adipose tissues in the periphery. GABRA5 LHA are a distinct subpopulation of GABAergic neurons and show decreased pacemaker firing in diet-induced obesity mouse models in males. Chemogenetic inhibition of GABRA5 LHA suppresses fat thermogenesis and increases weight gain, whereas gene silencing of GABRA5 in LHA decreases weight gain. In the diet-induced obesity mouse model, GABRA5 LHA are tonically inhibited by nearby reactive astrocytes releasing GABA, which is synthesized by monoamine oxidase B (Maob). Gene silencing of astrocytic Maob in LHA facilitates fat thermogenesis and reduces weight gain significantly without affecting food intake, which is recapitulated by administration of a Maob inhibitor, KDS2010. We propose that firing of GABRA5 LHA suppresses fat accumulation and selective inhibition of astrocytic GABA is a molecular target for treating obesity.

GABA synthesis in astrocytes mediates tonic inhibition to regulate patho-physiological processes in various brain regions. Monoamine oxidase B (MAO-B) has been known to be the most important metabolic enzyme for synthesizing GABA from the putrescine degradation pathway. MAO-B converts N 1 -acetylputrescine to N 1 -acetyl-γ-aminobutyraldehyde and hydrogen peroxide (H 2 O 2 ). Putrescine acetyltransferase (PAT), also known as spermidine and spermine N 1 -acetyltransferase 1 (SAT1), has been thought to be a feasible candidate enzyme for converting putrescine to N 1 -acetylputrescine. However, it has not been rigorously investigated or determined whether PAT/SAT1 contributes to GABA synthesis in astrocytes. To investigate the contribution of PAT/SAT1 to GABA synthesis in astrocytes, we conducted sniffer patch and whole-cell patch experiments with gene silencing of PAT/SAT1 by Sat1 shRNA expression. Our results showed that the gene silencing of PAT/SAT1 significantly decreased the MAO-B-dependent GABA synthesis, which was induced by putrescine incubation, leading to decreased Ca 2+ -dependent release of GABA in vitro . Additionally, we found that, from the brain slice ex vivo , putrescine incubation induces tonic GABA inhibition in dentate gyrus granule cells, which can be inhibited by MAO-B inhibitor, selegiline. Consistent with our in vitro results, astrocytic gene silencing of PAT/SAT1 significantly reduced putrescine incubation-induced tonic GABA current, possibly by converting putrescine to N 1 -acetylputrescine, a substrate of MAO-B. Our findings emphasize a crucial role of PAT/SAT1 in MAO-B-dependent GABA synthesis in astrocytes.

Alzheimer’s disease (AD) is one of the foremost neurodegenerative diseases, characterized by beta-amyloid (Aβ) plaques and significant progressive memory loss. In AD, astrocytes are proposed to take up and clear Aβ plaques. However, how Aβ induces pathogenesis and memory impairment in AD remains elusive. We report that normal astrocytes show non-cyclic urea metabolism, whereas Aβ-treated astrocytes show switched-on urea cycle with upregulated enzymes and accumulated entering-metabolite aspartate, starting-substrate ammonia, end-product urea, and side-product putrescine. Gene silencing of astrocytic ornithine decarboxylase-1 (ODC1), facilitating ornithine-to-putrescine conversion, boosts urea cycle and eliminates aberrant putrescine and its toxic byproducts ammonia and H2O2 and its end product GABA to recover from reactive astrogliosis and memory impairment in AD. Our findings implicate that astrocytic urea cycle exerts opposing roles of beneficial Aβ detoxification and detrimental memory impairment in AD. We propose ODC1 inhibition as a promising therapeutic strategy for AD to facilitate removal of toxic molecules and prevent memory loss.

Expenditure 2 RUNNING TITLES: Gabra5 neurons facilitate energy expenditure 3 4 Moonsun Sa, Eun-Seon Yoo, Wuhyun Koh, Mingu Gordon Park, Hyun-Jun Jang, Yong Ryul 5 Yang, Jiwoon Lim, Woojin Won, Jea Kwon, Mridula Bhalla, Heeyoung An, Yejin Seong, Ki 6 Duk Park, Pann-Ghill Suh, Jong-Woo Sohn, C. Justin Lee 7 1 KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro, 8

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that represents a major cause of death in many countries. AD is characterized by profound memory loss, disruptions in thinking and reasoning, and changes in personality and behavior followed by malfunctions in various bodily systems. Although AD was first identified over 100 years ago, and tremendous efforts have been made to cure the disease, the precise mechanisms underlying the onset of AD remain unclear. The recent development of next-generation sequencing tools and bioinformatics has enabled us to investigate the role of genetics in the pathogenesis of AD. In this review, we discuss novel discoveries in this area, including the results of genome-wide association studies (GWAS) that have implicated a number of novel genes as risk factors, as well as the identification of epigenetic regulators strongly associated with the onset and progression of AD. We also review how genetic risk factors may interact with age-associated, progressive decreases in cognitive function in patients with AD.