*5.4. Microglia*

Microglial activation in the brain of AD patients [138] has been linked to the triggering of a neuroinflammatory response that promotes Aβ-containing plaque formation and neuronal degeneration [139]. However, more recent studies have revealed that microglial activation in AD is a heterogenous process, and in line with these observations, specific and well-differentiated subpopulations of microglia also seem to have a protective effect in the brain of AD patients [140]. Furthermore, it has been reported that the plasminogen activating system modulates the activation of microglia in AD [141] and that treatment with rtPA triggers the activation of the above-mentioned neuroprotective microglial phenotype [142]. In contrast, the role of uPA in microglial activation in AD has been less well studied. However, it has been reported that Aβ-treated human microglia upregulate uPA and uPAR [143] and that uPAR is a marker of microglial activation in the brain of AD patients [144].

## *5.5. Neurons*

The amyloid hypothesis of AD is a neurocentric model in which Aβ deposition leads to progressive tau hyperphosphorylation, synaptic dysfunction and neuronal loss. However, despite its importance and long-time acceptance, a growing body of experimental evidence suggests that not only neurons, but all cellular and non-cellular elements of the NVU, play key roles in the pathogenesis of this disease [145]. Independently of these considerations, knowledge gathered over the last 3 decades has resulted in a better understanding of the biochemical process that leads to the production and accumulation of Aβ. More specifically, the proteolytic processing of APP by α-secretase on the cell membrane generates soluble APP<sup>α</sup>, which has been implicated in neuronal plasticity and synaptogenesis [146]. However, those APP molecules that are not processed by α-secretase are endocytosed and cleaved by β-secretase 1 (BACE1) and γ-secretase to generate Aβ 1–40 and 1–42 peptides [147–149], which have a harmful effect on cell survival and synaptic structure and function [150,151]. Inasmuch as our understanding of this process has grown, it has led to the discovery of different therapeutic strategies to minimize Aβ deposition that have been successfully tested in animal models of AD [152], but unsuccessful [153] or only partially effective in AD patients [154].

The last three decades of research on the role of the plasminogen activating system in the pathophysiology of AD have focused almost exclusively on the ability of tPA and uPA to cleave Aβ deposits. However, recent studies have also discovered a role for uPA in the pathogenesis of synaptic dysfunction in AD that does not require the conversion of plasminogen into plasmin.

#### 5.5.1. Plasminogen Activators and the Formation of Aβ Deposits

A role for plasminogen activators in the pathogenesis of AD was suggested by in vitro studies showing that plasmin triggers α-secretase-induced cleavage of Aβ in lipid rafts [155] and cleaves insoluble Aβ fibrils [156,157]. This was followed by work revealing a reduction in the expression and activity of plasmin in AD brains [158], most likely due to a decrease in tPA activity [157]. These observations were contested by a different group of investigators that detected normal concentrations of plasminogen and plasmin in the brains of AD patients [159], and postulated that the reported decrease in plasmin was actually due to the disruption of lipid rafts by abnormal cholesterol metabolism in the neuronal membrane [160].

Most of the studies on the role of plasminogen activators on the formation of Aβ deposits have been performed with tPA. Hence, it has been found that insoluble Aβ activates tPA [161] and increases the expression of tPA mRNA in cerebral cortical neurons, purportedly as an attempt to trigger plasmin-induced cleavage of extracellular insoluble Aβ-containing plaques [158]. In discrepancy with these studies, in vivo studies with mouse models of AD have revealed that chronic elevation of Aβ actually decreases tPA activity by enhancing the inhibitory effect of PAI-1 on tPA, and that the intracerebral injection of Aβ causes neuronal degeneration in animals genetically deficient in either tPA or plasminogen [162].

More specifically, the expression of PAI-1 is increased in the cerebrospinal fluid [163] and the brains of AD patients [164]. The clinical relevance of these observations is underscored by experimental work indicating that the genetic deletion of PAI-1 in the brain of a murine model of AD reduces the deposition of Aβ by increasing tPA-induced plasminmediated cleavage of Aβ-containing plaques [164]. Together, these data have led to the proposal of a model in which increased PAI-1 activity in the brain of AD patients abrogates tPA-induced plasmin-triggered cleavage of Aβ deposits. In seeming contradiction with a protective role of tPA in AD, other studies have shown that this plasminogen activator actually mediates the neurotoxic effect of Aβ via ERK 12 activation [165]. The role of uPA on the formation of Aβ deposits has been less well studied. Nevertheless, it has been reported that Aβ increases the abundance of uPA mRNA [156], and that as described for tPA, uPA also induces plasmin-mediated cleavage of insoluble Aβ-containing extracellular plaques [166].

#### 5.5.2. Role of Plasminogen Activators in Synaptic Dysfunction in AD

The idea that the extracellular accumulation of insoluble Aβ peptides is the cause of the cognitive decline observed in AD patients [167] has been challenged by neuropathological and clinical studies indicating that the development of cognitive deficits in these individuals correlates with abnormalities in synaptic structure and function more than with the number of tangles and insoluble Aβ-containing plaques [168,169]. This concept is of significant translational importance, because synaptic dysfunction is an early event in the pathogenesis of AD that is amenable to therapeutic interventions to prevent its development.

Our knowledge of the synaptic role of Aβ has increased significantly during the last 30 years. Hence, we know that the production of Aβ increases during neuronal activity [150], and that while at low concentrations, soluble Aβ induces presynaptic facilitation, at high concentrations triggers post-synaptic depression [170] by decreasing the abundance of glutamatergic receptors in the postsynaptic density [161,171] and enhances the excitotoxic effect of glutamate by blocking its reuptake from the synaptic cleft [172]. Additionally, high concentrations of Aβ impair long-term potentiation (LTP) and enhance long-term depression (LTD) by blocking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptor function [173], and augmen<sup>t</sup> the excitotoxic effect of glutamate by blocking its reuptake from the synaptic cleft [172]. Importantly, increased soluble Aβ has been linked to synaptic depression and the disruption of neuronal network activity in the brains of AD patients [171]. Furthermore, it has been reported that uPA antagonizes the

harmful effect of Aβ on synaptic structure and function by a mechanism independent of its ability to trigger proteolytic cleavage of Aβ-containing plaques [68].

Recent studies have shown that cleavage of Aβ-containing plaques is not the only role of uPA in AD brains. Hence, it has been found that the expression of uPA, but not of its receptor (uPAR), is decreased in the synapses of AD patients and 5XFAD mice (express human APP with the Swedish (KM670/671NL), Florida (I716V) and London (V717I) mutations together with a mutant presenilin 1 (M146L, L286V) under the control of the murine Thy-1 promoter), by the ability of Aβ to halt the transcription of uPA mRNA in neurons but not in astrocytes [68]. The translational importance of these findings is supported by observations indicating that treatment with recombinant uPA abrogates the harmful effect of soluble Aβ on synaptic structure and function, via its ability to induce the expression of neuronal cadherin (NCAD). Remarkably, in contrast with the reported effect of tPA and uPA on the proteolytic cleavage of Aβ-containing plaques, the effect of uPA on the synapses of AD patients and animal models of AD does not require the generation of plasmin.

#### 5.5.3. Plasminogen Activators, Physical Activity and Alzheimer's Disease

Physical activity has a direct effect on the expression and activity of components of the plasminogen activating system. More specifically, 6 months of intensive physical activity increase the intravascular concentration of tPA and uPA, and this effect is accompanied by a substantial decrease in the levels of PAI-1 [15,174]. These observations are of significant importance when interpreted in the context of studies showing that physical activity decreases the risk of AD [175] and improves the attention span, memory and executive functioning of healthy individuals [176]. Thus, it is tempting to postulate that plasminogen activators mediate the protective effect of exercise on cognitive function and the risk of developing AD. However, although scientifically plausible, to this date there are no data to support this hypothesis.

#### *5.6. Plasminogen Activators in Parkinson's Disease*

Parkinsonism is a clinical syndrome characterized by bradykinesia, resting tremor, rigidity and postural and gait impairment. Most cases of parkinsonism are caused by Parkinson's disease (PD), which is a neurodegenerative disease that affects 3% of the population older than 65 years of age [177]. The neuropathological hallmarks of this disease are the loss of dopamine-containing neurons in the substantia nigra and the presence of α-synuclein-containing intracellular inclusions. The extracellular levels of α-synuclein depend not only on its release from neurons, but also on its removal by proteolytic degradation. It is unclear if α-synuclein induces the expression or activity of tPA or uPA. However, plasmin cleaves and degrades α-synuclein, and α-synuclein upregulates PAI-1 [178]. It has thus been proposed that an excess of PAI-1 in the brains of PD patients prevents plasmin-induced clearance of α-synuclein aggregates [179]. The translational relevance of these findings is supported by the fact that increased levels of PAI-1 have been linked to a worse clinical prognosis in PD patients [180]. Despite the importance of these observations, to this date it is unclear if tPA- or uPA-catalyzed plasmin generation plays a role in the pathogenesis of this disease.

#### *5.7. Plasminogen Activators in Amyotrophic Lateral Sclerosis (ALS)*

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by a progressive decline in motor function caused by weakness and spasticity without sensory loss. Knowledge on the role of plasminogen activators in the pathogenesis of ALS is still in its earlier stages. However, it has been reported that plasminogen from ALS patients, and recombinant tPA and plasmin, induce motoneuron degeneration in BALB/c mice [181]. Likewise, experimental work with G93 mice (with a SOD1 mutation linked to familial ALS) and samples from ALS patients revealed that the abundance of uPAR increased in the ventral horn of the spinal cord of ALS patients and G93 mice, along with enhanced

uPA-dependent plasminogen activation in advanced stages of this disease. Remarkably, treatment with an inhibitor of uPA prolonged survival in these animals [182].

**Author Contributions:** Writing, editing and funding acquisition, M.Y.; writing and investigation, Y.W.; writing—original draft preparation, C.M.-J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by National Institutes of Health Grant NS-NS091201 (to M.Y.) and VA MERIT Award IO1BX003441 (to M.Y.).

**Data Availability Statement:** Not Applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
