Pathological phenotypes of astrocytes in Alzheimer’s disease

Astrocytes are involved in various processes in the central nervous system (CNS). As the most abundant cell type in the CNS, astrocytes play an essential role in neuronal maintenance and support, synaptic activity, neuronal metabolism, and amyloid-beta (Aβ) clearance. Alzheimer’s disease (AD) is a neurodegenerative disorder associated with cognitive and behavioral impairment. The transformation of astrocytes is involved in various neurodegenerative diseases, such as AD. Since astrocytes have functional diversity and morphological and physiological heterogeneity in the CNS, AD-related astrocytes might show various pathological phenotypes during AD. Astrocytes developing pathological phenotypes could contribute to AD progression. In this review, we provide an overview of the pathological phenotypes of astrocytes in the context of AD, highlighting recent findings in human and mouse AD.


INTRODUCTION
Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common type of dementia that is characterized by memory loss and cognitive dysfunction [1][2][3] .The pathological hallmark of AD is the deposition of amyloid-beta (Aβ) plaques and the formation of neurofibrillary tangles (NFTs) that are composed of hyperphosphorylated tau protein 4,5 .Despite numerous studies, the pathophysiological mechanisms of AD are still not fully understood.In contrast, neuronal cell death is a known prominent pathological feature of AD 4,5 .There is a limited understanding of the changes in astrocytes that promote AD pathogenesis.
The diversity of astrocyte populations has been described in different brain regions, and these populations are classified based on morphological and functional features [6][7][8][9][10][11][12][13] .As two large morphological groups, fibrous and protoplasmic astrocytes are located in the white and gray matter of the brain, respectively.Furthermore, astrocytes are classified based on distinct morphological and functional features, such as synapse association 11,12 , membrane properties, Ca 2+ signaling 12 , and neuronal maturation 13 .
Recent studies have shown that the alteration of astrocytes is involved in the initiation and progression of AD 14,15 .As astrocytes play various physiological roles in synapse formation and function, neurotransmitter release and uptake, the production of trophic factors, and neuronal survival by energetic supports 6,[16][17][18][19] , the morphological and functional dysregulation of astrocytes is linked to neuronal cell death in AD 20,21 .Understanding AD-related astrocyte subtypes could help identify new pathophysiological mechanisms of AD.In this review, we explore the features of ADrelated astrocytes and discuss the pathological phenotypes of astrocytes in the context of humans with AD and mouse AD models.
The reactive phenotype of astrocytes in AD Proinflammatory, neurotoxic A1 reactive astrocytes.Astrocytes can be changed to reactive astrocytes via morphological, molecular, and functional alterations in various pathological conditions 22,23 .Reactive astrocytes induce neuropathology and neurodegeneration in neurodegenerative diseases and neurotoxic conditions 24 .As part of astrocyte polarization, reactive astrocytes can switch to either the pro-inflammatory, neurotoxic A1 phenotype (A1 astrocytes) or the anti-inflammatory, neuroprotective A2 phenotype (A2 astrocytes) 25,26 .
As a significant phenotype of reactive astrocytes in AD, A1 reactive astrocytes were identified in the brains of AD patients 28 .A1 reactive astrocytes have high gene levels of glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B (S100B), and complement C3 (C3) in the brains of AD patients 28 .
The death phenotypes of astrocytes in AD Apoptotic phenotype of astrocytes.Apoptosis is involved in various diseases of the nervous system 32 .The presence of numerous apoptotic cells is a pathological feature of the brains of patients with AD 33,34 .The levels of the active form of caspase-3 protein, an executioner caspase in apoptosis, were increased in the brains of AD patients 33 .The apoptosis of astrocytes may contribute to the pathogenesis of AD.
In a study on the apoptotic phenotype of astrocytes in the brains of AD patients, the number of DNA fragmentationpositive astrocytes was increased in the temporal lobe of the brain 34 .The number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic astrocytes was increased in the brains of AD 35 .The density of TUNELpositive astrocytes was correlated with the density of uncored and cored senile plaques, which are polymorphous Aβ protein deposits 35 .TUNEL-positive apoptotic astrocytes showed regressive changes with fragmented processes and cytoplasmic vacuoles in the brains of AD patients 36 .Figure 1 summarizes the apoptotic phenotype of astrocytes in human AD.
Ferroptosis phenotype of astrocytes.Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron that is morphologically, biochemically, and genetically distinct from other forms of cell death, including apoptosis, necrosis, and autophagy 37 .Ferroptosis is involved in neuronal death during AD pathogenesis 38,39 .Ferroptosis of astrocytes has been identified in AD 38,39 .
In a study on the ferroptosis of astrocytes in AD patients, ferroptosis-related oxidative stress markers, including 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), were elevated in astrocytes of the cerebral cortex of brains of AD patients 38 .The number of ferroptotic astrocytes was increased in the brains of AD patients 38 .As an upstream molecule involved in ferroptosis in astrocytes, the levels of NADPH oxidase 4 (NOX4) were increased in the 4-HNE-positive astrocytes in the cerebral cortex of brains of AD patients 38 .
In a study of ferroptosis in AD model mice, the levels of the ferroptosis-related proteins 4-HNE and MDA was elevated astrocytes in the cortex of APP/PS1 AD model mice 38 .Furthermore, the number of ferroptotic astrocytes was increased in the cortex of APP/PS1 AD model mice 38 .The expression of ferroptosis-related genes, such as iron response element binding protein 2 (Ireb2), citrate synthase (Cs), ribosomal protein L8 (Rpl8), and prostaglandin-endoperoxide synthase 2 (Ptgs2), was upregulated in hippocampal tissues of APP/ PS1 AD model mice 39 .Figure 1 summarizes the phenotypes of ferroptotic astrocytes in human and mouse AD.

The senescence phenotype of astrocytes in AD
Aging is a primary risk factor in the pathogenesis of AD 40 .Cellular senescence is the hallmark of aging 41,42 .Cellular senescence in the brain may link the aging process to AD progression.The senescence of astrocytes is related to AD pathogenesis 43 .
In a study on the senescence of astrocytes in AD patients, senescent astrocytes showed upregulated expression of cyclindependent kinase inhibitor 2 A (CDKN2A) (also known as p16INK4a), which is a marker of senescence, in the frontal cortex of AD patients 44 .The number of senescent astrocytes was increased in AD patients 44 .As a marker of aging-related DNA damage, the expression of γH2AX, a phosphorylated form of H2A.X variant histone (H2AX), which is a part of the nucleosome structure, was increased in the hippocampal regions and cerebral cortex of AD patients 45 .γH2AX is produced through phosphorylation in response to the formation of double-stranded breaks in chromosomal DNA [46][47][48] .Furthermore, the phosphorylation of H2AX induces the translocation of phosphatidylserine to the outer cell membrane and the internucleosomal DNA fragments during apoptosis 49 .
In a study on the senescence of astrocytes in mouse AD, the expression of senescence-associated genes such as Cdkn2a was increased in the astrocytes of tau MAPT P301S PS19 transgenic mice 44 .Pharmacological elimination of senescent astrocytes by the senolytic agent ABT263 prevented the upregulation of senescence-associated gene expression and attenuated tau phosphorylation in the brains of tau MAPT P301S PS19 transgenic mice [50][51][52] .Figure 1 summarizes the phenotypes of senescent astrocytes in human and mouse AD.
The functional impairment of astrocytes in AD Impaired Ca 2+ signaling function in astrocytes during AD.Astrocytes play a role in maintaining the homeostasis of neuronal circuits and regulating neuronal activity via intracellular calcium signals 6,53,54 .
Dysfunction of astrocyte calcium signaling leads to network hyperactivity in the early stage of AD 55 .Dysregulation of calcium signaling in astrocytes might contribute to the pathological progression of AD.
In the study of astrocyte calcium signaling in AD patients, the expression of inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) (also known as IP3 receptor type 2 (IP3R2)), an intracellular calcium release channel, was decreased in the astrocytes in brains of AD patients 55,56 .Furthermore, the levels and nuclear localization of nuclear factor of activated T cells 3 (NFAT3), which regulates calcineurin (CN), a Ca 2+ /calmodulin-dependent protein phosphatase, were elevated in the brains of AD patients 57 .Elevation of CN in astrocytes triggered the induction of genes associated with inflammatory responses in early-stage AD patients 58 .
In a study on calcium signaling in astrocytes in mouse AD, decreased calcium signaling was caused by a reduction in Itpr2 expression in astrocytes before the accumulation of Aβ plaques in the early stage of AD in App NL-F model mice 55,56 .Figure 1 summarizes the phenotypes of Ca 2+ signaling-impaired astrocytes in human and mouse AD.
In a study on glutamate uptake in astrocytes in AD patients, glutamate transport activity was reduced in the cortex of brains of AD patients 72 .The expression of the EAAT2 protein and glutamate transport activity were decreased in the frontal cortex of AD patients 73 .Additionally, a reduction in glutamate transport activity was associated with enhanced Aβ accumulation in AD patients 73 .
In a study on glutamate uptake in the astrocytes of AD mice, the loss of Slc1a2 exacerbated cognitive impairment in the AD model mice 74 .Additionally, restoration of Slc1a2 function improved cognitive functions, restored synaptic integrity and reduced amyloid plaques in a AD model mice 75 .Figure 1 summarizes the phenotypes of impaired glutamate buffering in astrocytes in human and mouse AD.

CONCLUSIONS
We reviewed the pathological phenotypes of astrocytes in AD and discussed the transcriptomic and proteomic features of the pathological phenotypes of astrocytes in the brains of AD patients and AD model mice.We described the reactive phenotype, death phenotype, senescence phenotype, and functional impairment phenotypes of astrocytes in human and mouse AD.The development of pathological phenotypes by astrocytes may be an essential event in the pathogenesis of AD.Understanding the pathological phenotypes of astrocytes may help maintain normal brain function and prevent neurodegeneration during AD.Along with current therapies for AD that target Aβ and tau pathology, the proper control of astrocyte pathology could be an alternative therapeutic approach for AD treatment.