Neuroglial Senescence, α-Synucleinopathy, And The Therapeutic Potential Of Senolytics in Parkinson's Disease Part 2

Astrocytic α-Synucleinopathy in Parkinson's Disease

Astrocytes are the most abundant cell type in the CNS, constituting an estimated 20–40% of the mammalian brain and are about five times as prevalent as neurons (Khakh and Sofroniew, 2015; Troncoso-Escudero et al., 2018; Giovannoni and Quintana, 2020). 

Astrocytes are cells that play an important role in the brain. They are closely connected with neurons to form the famous "glial network" and participate in various functions of the brain. In recent years, many studies have shown that astrocytes are closely related to memory, affecting various behaviors and ways of thinking in our lives.

First, astrocytes facilitate communication between neurons. Neurons transmit information through synapses, and the synaptic structures of astrocytes and neurons are very close. As a result, they enhance communication between neurons, helping us better process information and memory.

Secondly, astrocytes can remove metabolic waste products produced during neuronal activity. The activity of neurons produces large amounts of waste products such as lactic acid and ammonia. If these wastes cannot be removed in time, they will accumulate in the brain, affecting the normal activity of neurons and leading to memory decline. Astrocytes can remove waste and help us maintain good memory.

In addition, astrocytes can also produce neurotrophic factors, such as BDNF. These factors can promote connections between neurons and enhance memory. Studies have shown that BDNF secreted by astrocytes is closely related to memory and learning, and they can promote the proliferation of neurons and the formation of synapses.

In summary, astrocytes are inextricably linked to memory. They can promote communication between neurons, remove metabolic waste, and produce neurotrophic factors, which help us maintain good memory and learning abilities. Therefore, we should focus on protecting the health of brain cells and maintaining a positive lifestyle so that our brains can work healthily and happily. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thus enhancing the connectivity and function of neural networks. These effects can help improve memory, learning, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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Over twenty different structural and functional subpopulations of astrocytes exist in the adult human CNS (Miller, 2018; Verkhratsky and Nedergaard, 2018). Despite their heterogeneity, the overarching function of astrocytes is to provide support for neurons and to help ensure a homeostatic environment in the brain. Toward this end, astrocytes perform many functions. 

For example, they contribute to BBB maintenance, promote normal synaptic function, secrete neurotrophic molecules, modulate their microenvironment as necessary, and are involved with the regulation of neurogenesis, lipoprotein secretion, and cerebral blood flow (Ding et al., 2021). Additionally, in experimental mouse models with either ablated or dysfunctional microglia, astrocytes became activated and phagocytic to help compensate for the microglial loss (Konishi et al., 2020). Even in their reactive state, astrocytes exhibit functional variability. 

From rest (A0), astrocytes can enter one of two polarized forms of activation based on their gene expression profile in response to stimuli: A1 or A2. In broad terms, A1 is neurotoxic and A2 is neuroprotective (Zamanian et al., 2012; Liddelow and Barres, 2017; Ding et al., 2021). Astrocytes and microglia share a close relationship regarding reactivity and phagocytosis. Simply stated, reactive microglia give rise to astrocyte reactivity (Liddelow et al., 2017). 

Indeed, the cytokines Il-1α, TNF-α, and C1q, which are released from reactive M1 microglia during chronic neurodegenerative conditions, are both necessary and sufficient for producing an A1 phenotype in astrocytes (Liddelow et al., 2017). Blocking the ability of microglia to activate A1 astrocytes helps to preserve in vivo dopaminergic neuron viability (Hinkle et al., 2019). 

A1 reactive astrocytes contribute to PD pathology by secreting a host of neurotoxic and pro-inflammatory factors that inhibit synaptogenesis and promote dopaminergic neuronal death (Liddelow and Barres, 2017). 

Conversely, in response to traumatic or ischemic CNS injury, M2 microglia induce the genetic upregulation and downregulation of astrocytes to exert a neuroprotective A2 phenotype (Hernández et al., 2021). A2 astrocytes become reactive in response to microglial secreted IL-1β, IL-6, and nuclear factor IA (NFIA) as well as the silencing of miR-21 (Su et al., 2019; Tchieu et al., 2019). 

The A1 astrocyte phenotype has been observed in cultured astrocytes treated with α-synuclein aggregates (Lee et al., 2010; Troncoso-Escudero et al., 2018). Furthermore, cultured astrocytes readily take up secreted α-synuclein aggregates directly from neighboring co-cultured neurons to adopt an A1 phenotype (Lee et al., 2010). 

Neuronally secreted α-synuclein binds to the toll-like receptors (TLRs) of astrocytes and promotes an increased neuroinflammatory response through activated TLR signaling (Lee et al., 2010; Fellner et al., 2013; Rannikko et al., 2015; Booth et al., 2017; Verkhratsky and Nedergaard, 2018). 

The direct relationship between α-synuclein aggregates and reactive A1 astrocytes has also been observed in vivo, through a transgenic mouse model that expressed human α-synuclein (Lee et al., 2010). Additionally, in postmortem, PD patient brains, inclusions of αsynuclein aggregates have been found in astrocytes as well as in neurons (Wakabayashi et al., 2000; Hishikawa et al., 2001; Braak et al., 2007; Booth et al., 2017). 

When mutated α-synuclein is selectively expressed in mouse astrocytes, the astrocytes develop the A1 phenotype, neuroinflammation increases, microglia become activated and mice develop paralysis (Gu et al., 2010). 

Despite the detrimental effects of reactive astrocytes induced by α-synuclein, astrocytes also contribute to the degradation and removal of mutated α-synuclein and are more efficient at it than neurons are (Tsunemi et al., 2020).

BRAIN CELLULAR SENESCENCE IN PARKINSON'S DISEASE

Introduction to Cellular Senescence

Three fates ultimately await very aged or damaged cells: senescence, apoptosis, or autophagy (Vicencio et al., 2008). Autophagy (self-eating) is largely a homeostatic mechanism through lysosomal destruction of old or damaged cellular components, allowing for the recycling of cellular material. However, recent evidence also supports the view that autophagy plays an important role in mammalian cell death (Jung et al., 2020). 

Apoptosis (self-killing) is a morphologically unique, genetically programmed cell death. It has important roles in development, aging, and atrophy, to maintain an appropriate level of cells (Elmore, 2007). It also has many critical roles in immune system function (Nagata and Tanaka, 2017). 

Cells can also enter a state of senescence (stable cell cycle arrest) as a homeostatic response to various stressors to mitigate the proliferation of damaged cells and prevent neoplastic transformation (Kritsilis et al., 2018). 

Senescent cells are metabolically active, stable, and viable, unlike cells destined for death and recycling. The classical understanding is that only mitotically active cells in the periphery can enter senescence by becoming arrested in the G1 phase, unlike quiescent cells that arrest in G0 (Di Leonardo et al., 1994; Vicencio et al., 2008).

However, post-mitotic cells in the central nervous system are now recognized as being able to undergo senescence (Baker and Petersen, 2018). There is some evidence of crosstalk and shared regulated pathways between these three cell fate states, but there is much that is currently not understood about their relationships with one another (Abate et al., 2020). 

Also, there is some debate currently over the post-mitotic status of glia and CNS neurons. Senescent cells accumulate during aging, both from an increased rate of production and a decreased clearance rate (Karin and Alon, 2021). Although the accumulation of senescent cells is associated with increasing age, it is not age-dependent. Senescence is a dynamic and context-specific state that involves many biochemical pathways, such as the p53/p21WAF1/CIP1 and p16INK4A/pRB tumor suppression pathways (McConnell et al., 1998; Kumari and Jat, 2021). 

Senescent cells that form in response to stress during earlier life stages, such as from oncogene activation, inflammation, or DNA damage, contribute to protective functions such as tumor suppression, wound healing, preventing the propagation of tissue damage, and embryogenesis (Storer and Keyes, 2014; Ovadya and Krizhanovsky, 2018). 

In non-pathological states, senescent cells attract the immune system and are cleared (Langhi Prata et al., 2018). There is a physiological threshold where senescent cells interfere with their clearance, described by the recently proposed "immune threshold theory of senescent cell burden" (Tchkonia et al., 2021). 

According to this theory, once the saturation of senescent cells passes the senescent cell abundance threshold, the spread of cellular senescence outpaces the immune system's ability to clear them and cellular senescence becomes "self-amplifying" and contributes to age-associated diseases (Tchkonia et al., 2021). 

The age-associated nature of senescent cell accumulation and the threshold theory of senescent cell burden make it a clear example of evolutionary antagonistic pleiotropy (Mitteldorf, 2019). 

Senescent cells have characteristic phenotypes marked by irreversible and permanent cell cycle arrest, SASP, disruption of normal mitochondrial form and function, metabolic changes, genomic DNA damage, telomere attrition, altered epigenetics, impaired DNA repair mechanisms, altered proteostasis, increased reactive oxygen species (ROS) production and increased ROS-mediated damage (Martínez-Cué and Rueda, 2020). 

The phenotypical changes that senescent cells undergo allow the cell to influence its immediate environment through SASP as well as to ensure cell cycle arrest (Birch and Gil, 2020). The hypersecretory phenotype SASP is a key hallmark of senescent cells. 

The composition of SASP includes pro-inflammatory cytokines such as IL-6 and IL-8 as well as insulin-like growth factors IGFBP3, IGFBP4, and IGFB7, which have pro-senescence and tumor-suppressant properties (Özcan et al., 2016; Soto-Gamez and Demaria, 2017). In addition to cytokines and growth factors, SASP also includes chemokines, matrix-metalloproteinases, and many other constituents (Cuollo et al., 2020). 

The exact composition of SASP is dynamic and heterogeneous depending on the senescent cell type, driver of senescence, and cellular context (Coppé et al., 2011; Maciel-Barón et al., 2016; Birch and Gil, 2020). 

Senescence-associated secretory phenotype works to reinforce the cellular senescence of its source through the autocrine senescence process as well as spreading senescence to nonsenescent neighboring cells via paracrine senescence (Hoare and Narita, 2013; Borodkina et al., 2018). SASP can be activated in various ways, but it has been argued that DNA damage is the main upstream driver (Kumari and Jat, 2021). 

The NF-κB and JAK2/STAT1 pathways are the main regulators of SASP (Gao et al., 2021). 

Therefore, SASP expression is influenced by pathways that modulate the NF-κB and JAK2/STAT1 pathways, such as the mammalian target of rapamycin (mTOR) pathway, mitogen-activated protein kinase (MAPK) signaling, the phosphoinositide 3 kinase (PI3K) pathway, the DNA damage response (DDR), and GATA4/p62-mediated autophagy (SotoGamez and Demaria, 2017; Birch and Gil, 2020).

Microglial Senescence in Parkinson's Disease

Senescent microglia differ from activated microglia. Microglia undergo non-pathologic changes related to advanced age that bring them to a senescent state, sometimes synonymously referred to as dystrophy, although dystrophy typically only refers to the morphological changes of senescent microglia (Streit et al., 2004, 2009, 2014; Angelova and Brown, 2019; Shahidehpour et al., 2021). 

Dramatic morphological dystrophy of senescent microglia is not seen in the brains of mice but is observed in humans (Streit et al., 2014). 

Senescent microglia undergo distinct age-related morphological, functional, and distribution changes (Streit et al., 2014; Shaerzadeh et al., 2020; Brawek et al., 2021). For example, senescent microglia experience swelled spherical somas, a fragmented and beaded cytoplasm, and reduced ramification (very shortened processes, a reduction in the number of processes, and a reduction in the number of branches per process) (Shaerzadeh et al., 2020; Brawek et al., 2021). 

Senescent microglia also experience a change in their uniform distribution resulting in increased location-specific density (Sikora et al., 2021). 

The total number also increases with age, although this seems debatable (Kodama and Gan, 2019; Ritzel et al., 2019; Sikora et al., 2021). Functionally, senescent microglia resemble a mild activation state (Lopes et al., 2008; Streit et al., 2014; Rodriguez et al., 2015). 

For example, there is an increased proportion of senescent microglia in the aged brain that experience spontaneous Ca2+ transients compared to middle-aged or young brains in mice (Del Moral et al., 2019). 

Neurodegenerative insult still elicits an attempt by aged microglia to boost their heightened baseline activation levels. For example, aged mouse models of Alzheimer's disease experience a higher degree of intracellular Ca2+ transients in senescent microglia than what is seen due to aging alone (Brawek et al., 2014). 

Additionally, senescent microglia have a reduced rate of migration to injured areas and a reduced rate of process movement toward injury (Wong, 2013; Brawek et al., 2021). Furthermore, aged mice have impaired microglial phagocytic ability (Ritzel et al., 2019). As the proportion of senescent microglia accumulates with age or age-related disease, they eventually outnumber healthy microglia and produce an enhanced neuroinflammatory response to injury, compared to young brains (Sugama et al., 2003; Wasserman and Schlichter, 2008; Luo et al., 2010). 

Together, these observations support the notion of impaired function of senescent microglia in aged brains. As the brain ages, it experiences chronic low-level oxidative stress and subsequent inflammation (Streit et al., 2014). 

Aging drives the switch from neuroprotective microglia to senescent microglia, as well as an increased concentration of senescent microglia in the SNpc (Angelova and Brown, 2019). As part of the age-related inflammatory milieu of the CNS, senescent microglia chronically secrete pro-inflammatory cytokines such as IL-6, IL-8, IL-1β, and TNF-α (Sierra et al., 2007; Wong, 2013; Sikora et al., 2021). 

They also release a reduced amount of anti-inflammation cytokines and increased levels of ROS, which are Nox-2 dependent (Angelova and Brown, 2019; Geng et al., 2020). 

The pro-inflammatory secretions of senescent microglia are akin to the pro-inflammatory secretions of activated microglia. However, consistent with a senescent state, senescent microglia also have genomic and mitochondrial DNA damage as well as telomere shortening (Costa et al., 2021; Hsiao et al., 2021). 

Senescent microglia also have upregulated expression of senescent markers such as Bcl-2, senescence-associated βgalactosidase (SA-β-gal), p16INK4a, p21WAF1/CIP, lipofuscin, and H2AX[pS139] (Ritzel et al., 2019). The senescent microglial response to injury is delayed and prolonged, which helps to shape the chronic and slowly progressive nature of PD (Luo et al., 2010; Damani et al., 2011). 

The significance of an aged CNS with senescent microglia in PD pathology is demonstrated clearly in mouse models. For example, a low dose of the pesticide rotenone reduced the number of SNpc dopaminergic neurons by up to 30% in old rats but was benign when given to young rats (Phinney et al., 2006). 

Similarly, old mice with age-related senescent microglia experienced a markedly more severe reduction of dopaminergic neuron concentration in response to MPTP injections than neonatal mice (Sawada et al., 2007). Despite the value of toxin rodent models in the study of PD, it is noted that there are some limitations in their applicability (Harms et al., 2021). 

Parkinson's disease has been epidemiologically linked to iron exposure (Angelova and Brown, 2019). Analysis of SNpc tissue from postmortem PD patients also reveals an elevated iron concentration compared to controls (Angelova and Brown, 2019). 

Despite not being the main cell type to store iron in the brain, senescent microglia have increased levels of ferritin, which increases their internal exposure to iron-related oxidative stress (Lopes et al., 2008; Angelova and Brown, 2019; Galaris et al., 2019). 

Oxidative stress in microglia, whether due to heightened iron levels or catabolizing degenerative neurons that contain iron-rich neuromelanin, prompts senescent microglial to secrete pro-inflammatory factors. Therefore, in addition to aging and α-synucleinopathy, intracellular iron contributes to the senescent secretome. There are morphological and physiological differences between male and female microglia. 

The sex-related differences in microglia correspond to the sex differences in PD risk. Men have between 1.5× and 2× the risk for developing PD than females (Wooten et al., 2004; Cerri et al., 2019). Furthermore, estrogens are neuroprotective against PD (Lee et al., 2019). 

The sex differences in microglia are thought to be the result of estrogen priming (Cerri et al., 2019). For example, there is evidence that the female 6-OHDA mouse model of PD has higher levels of estrogens than males, which prompts their activated microglia to polarize toward the neuroprotective M2 phenotype rather than the male-dominant pro-inflammatory M1 phenotype (Siani et al., 2017). 

The sex differences in brain inflammation response, PD risk, and microglial physiology due to estrogen activity have also been confirmed in humans (Hanamsagar et al., 2017; Villa et al., 2018; Acosta-Martínez, 2020).

Astrocytic Senescence in Parkinson's Disease

Senescence and reactivity are distinct astrocytic cell fates, yet they share some common features. A recent transcriptomic study revealed a wide range of senescent-related markers upregulated in senescent astrocytes such as SA-β-gal, IL6, IL8, IL1A, IL1B, CDKN1A, the p53/p21WAF1 and p16INK4A/pRB pathways, CYR61, CCND1, IGFNP5, and IGFBP2 (Simmnacher et al., 2020). 

Senescent astrocytes also experience an upregulation of high mobility group B (HMGB) proteins, increased production of vimentin and glial fibrillary acidic protein (GFAP), in addition to a reduced expression of neurotrophic factors and nuclear lamina protein laminB1 (Han et al., 2020). Some of the upregulated inflammatory marker genes are shared between reactive and senescent astrocytes. 

For example, astrocytes in both states experience upregulated pro-inflammatory cytokines, chemokines, proteases, and growth factors (Maciel-Barón et al., 2016; Cohen and Torres, 2019; Simmnacher et al., 2020). The morphology of senescent astrocytes changes to become flattened, and enlarged, and have vacuolized lysosomes (Bitto et al., 2010; Cohen and Torres, 2019). 

Normal aging gives rise to astrocytic senescence-inducing factors such as damaged DNA and shortened telomeres (Bhat et al., 2012; Kang et al., 2015). Senescent astrocyte accumulation also occurs in neurodegenerative diseases (Limbad et al., 2020). Compared to control tissue, the SNpc tissue from five postmortem PD patients exhibited a large increase in the levels of p16INK4a and the SASP components MMP-3, IL-6, IL1α, and IL-8, as well as the reduced nuclear level of lamin B1 (Freund et al., 2012; Chinta et al., 2018). 

Interestingly, the drop in nuclear lamin B1 levels was only observed in astrocytes, whereas nuclear lamin B1 levels remained unchanged in the astrocyte neighboring tissues between the PD SNpc and control tissues (Chinta et al., 2018). 

These results suggest that astrocytes have a uniquely elevated susceptibility to becoming senescent in PD. Furthermore, multiple studies have shown that cultured human and rodent astrocytes are more susceptible to toxin-induced senescence than fibroblasts (Bitto et al., 2010; Chinta et al., 2018). 

However, one study showed that neurons in long-term cultures of primary rat cortical cells became senescent before neuroglia did (Moreno-blas et al., 2019). It is thought that this aberrant result was due to dysfunctional age-associated autophagy (Morenoblas et al., 2019). 

Senescent astrocytes also have decreased expression of glutamate transporters, and therefore, promote glutamate toxicity and subsequent death of surrounding neurons (Limbad et al., 2020). Paraquat has been a widely used toxic herbicide and has a chemical structure similar to the dopaminergic neurotoxin MPTP (Wang et al., 2017). 

Whether chronic occupational exposure to paraquat is causal for PD in humans is debated. Although the connection between paraquat and PD in humans has seemed strong in the past, the more current perspective considers the correlation to be weak (Weed, 2021). Regardless, paraquat has been successfully used for animal models of PD, replicating important hallmarks of the disease such as increased α-synuclein levels and α-synuclein aggregations in SNpc neurons, leading to dopaminergic neuron loss and movement impairment (Cristóvão et al., 2020). 

hiPSCs that have been differentiated into astrocytes and exposed to paraquat cease to proliferate and have increased levels of SA-β-gal, p16INK4a, and IL6, indicating a senescent state (Chinta et al., 2018). The hiPSC-derived astrocytes also had an increased number of 53BP1 foci due to the upregulation of DNA damage signaling, which stimulates SASP expression (Chinta et al., 2018). 

Other environmentally toxic chemicals that have been linked to PD, like the pesticide rotenone and 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), have also been shown to cause premature senescence in human astrocytes in a dose-dependent manner (Wan et al., 2014; González-Barbosa et al., 2017; Simmnacher et al., 2020). 

The oxidative stress and subsequent inflammation associated with neurodegenerative diseases, non-pathological advanced age, and environmental toxin exposure drive elevated senescent astrocyte levels (Bitto et al., 2010; Si et al., 2021). 

For example, the hormone angiotensin II has been implicated in producing intracellular free radicals, increasing oxidative stress, promoting mitochondrial dysfunction, and accelerating inflammation related to aging (Benigni et al., 2010). 

Angiotensin II has also been shown to cause senescence in cultured human astrocytes in a concentration-dependent manner by producing superoxide oxidative stress (Liu et al., 2011). The transcriptome of fetal human astrocytes that have been made senescent by transient oxidative stress exposure has been characterized. 

Unsurprisingly, genes associated with neural development and differentiation were downregulated as well as some genes related to injury response, whereas pro-inflammatory genes were upregulated (Crowe et al., 2016).

INTERACTIONS BETWEEN α-SYNUCLEINOPATHY AND NEUROGLIAL SENESCENCE IN PARKINSON'S DISEASE

A Self-Amplifying and Vicious Cycle

The physiological connections between senescent neuroglia and the development of neuronal α-synucleinopathy are feed-forward oxidative stress and inflammation cascades. 

The brain is a highly metabolically active organ and consumes about 20% of the basal oxygen in humans (Cobley et al., 2018). Free radicals are abundant in the brain and are necessary for the delicate redox signaling that is critical for many CNS functions (Cobley et al., 2018; Franco and Vargas, 2018; van Leeuwen et al., 2020). However, when the oxidative balance favors cellular stress, cellular damage results. 

According to the free radical theory of aging, a consequence of aerobic metabolism over the lifespan is oxidative damage that results in the aging process (Harman, 1956). Central nervous system oxidative stress has been shown to induce neuroglial senescence. 

Specific oxidative stress effects on neuroglia that lead to a senescent state include morphological changes, disrupted mitochondrial function, altered cellular signaling, altered cellular metabolism, damaged DNA, shortened telomeres, altered chromatin structure, altered proteostasis, and impaired DDR (Correia-Melo and Passos, 2015; Vasileiou et al., 2019; Martínez-Cué and Rueda, 2020). 

The pro-inflammatory SASP of senescent astrocytes and microglia contribute to the low-grade chronic inflammation that is associated with CNS aging (Norden and Godbout, 2013; Streit et al., 2014; von Bernhardi et al., 2015; Martínez-Cué and Rueda, 2020). CNS inflammation contributes to the α-synuclein toxicity that drives α-synucleinopathy in PD. 

Neuroinflammation is increasingly being recognized as one of the major contributors to PD pathology rather than just a by-product of the disease process itself (Harms et al., 2021; Hirsch and Standaert, 2021). Neuroinflammation in PD involves both the innate immune system and the adaptive immune system since PD-related BBB compromise facilitates the entrance of peripheral immune cells into the CNS (de Vries et al., 2012; Cardinale et al., 2021). 

The two Nod-like receptor (NLR) proteins, NLRP3 and NLRC4, are part of the innate immune system, are inflammasome activators in microglia and astrocytes, and activate caspase-1 (Freeman et al., 2017). 

NLRP3 activation in microglia by ROS leads to the release of the pro-inflammatory cytokines IL-1β and IL-18 (Kam et al., 2020). ROS can also activate NLRC4 in astrocytes, prompting the release of IL-1β and IL-18 as well (Lim et al., 2019).

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