The other type of lesion that is prominent in SPMS besides cortical lesions are smoldering lesions. These are focal white matter lesions that occur in RRMS that slowly and gradually expand in SPMS. In the CNS of MS patients complement deposits occur in pattern II demyelinating lesions on degenerating myelin sheaths along the active lesion rim. Cortical lesions are pattern III lesion without complement deposits that follow a perivascular distribution. In MS, pattern II lesions are often in the periventricular areas, cerebellum, brainstem, optic nerves, and in the cervical and thoracic spinal cord.
Microglia can directly release high amounts of neurotoxic factors like ROS/RNS in SPMS that can cause neurodegeneration. The main enzyme for ROS production in microglia is NADPH oxidase. ROS/RNS can also induce mitochondrial dysfunction in neurons that persists and accumulates over time. In SPMS, deep cortical neurons contain mutations in mitochondrial DNA. MS lesions have significant mitochondria disturbances including decreased expression of electron transport chain complex I, III and IV, which correlates with axon damage. The energy deficiencies due to mitochondrial dysfunction amplify OS via the release of more oxygen radicals into the CNS. Considering the high energy consumption the brain utilizes, impaired energy production due to mitochondrial dysfunction likely contributes to neurodegeneration.
A senescent type of microglia lines the rims of chronic active iron rimmed lesions (IRLs) “smoldering lesions”. They slowly expand over time in SPMS, compared to non IRLs that shrink. They also occur in absence of peripherally mediated inflammation.
These microglia are chronically activated and highly release pro-inflammatory cytokines, produce ROS/RNS, and elevate iron storage. They also decrease phagocytic activity and motility which causes myelin debris to accumulate. This induces a pro-inflammatory environment along the rims of chronic active lesions.
Microglia reactivity also increases with disease duration and associates with more diffuse NAWM injury like myelin loss and axon damage in SPMS. Clusters of reactive microglia “microglia nodules” are seen in areas around lesions and in NAWM in SPMS. They associate with Wallerian degeneration in early MS lesions that occur before demyelination. ROS/RNS produced by microglia directly damage neurons via loss of cytochrome C oxidase (COX1), and mitochondrial respiratory chain complex IV activity, causing mitochondrial dysfunction.
The elevated iron is from the myelin debris they have phagocytized that highly contributes to their senescence. HO-1 mobilizes iron from heme in the debris and increases expression in microglia in SPMS. The iron is stored safely in ferritin in their cytosol and increases as MS progresses.
Ferritin is shuttled in the form of divalent cations (Fe3+) to autophagosomes by ‘nuclear receptor coactivator 4’ (NCOA4), which upregulates in SPMS. This process releases harmful, redox active ferrous iron in the form of divalent cations (Fe2+). These react with H2O2 that is naturally produced by mitochondria respiration which amplifies oxidative injury via the Fenton reaction that generates highly toxic hydroxyl (OH) radicals. Fe2+ uptake by activated microglia can fragment and degenerate them, releasing it again and damaging surrounding tissue, axons, and neurons. Ferrous iron can also induce toxicity indirectly as seen by FeSO4 (a Fe2+ donor) highly increasing ROS. These changes occur along with reductions in the antioxidant pathway (system xCT, glutathione peroxidase 4 and glutathione), that increases lipid peroxidation. Replacing oxidized FAs at the sn-2 position of membrane phospholipids are targets for lipid peroxidation and ferroptosis. The enzymes ACSL4 and LPCAT3 incorporate arachidonic acid into membrane phospholipids. They increase in SPMS to repair oxidative damage to cell membranes, but are rapidly peroxidized. Membrane phospholipids lipid peroxidation in plasma membranes, organelles, and myelin generates highly toxic 4-hydroxy-2-nonenal (4-HNE). OL ferroptosis and slow lesion expansion. It highly increases in SPMS in areas surrounding axons that suggest lipid peroxidation in OLs and myelin sheaths at lesion rims. When these occur with reduced antioxidant GSH levels, that occurs with age and disease, the programmed cell death pathway ferroptosis occurs.
This microglia phenotype, has altered surveillance with less dendritic branching and mobility, impaired phagocytosis, and a sustained inflammatory response to damage. These may cause them to aberrantly secrete and activate complement proteins They are termed ‘damage associated microglia’ (DAM) and are seen in neurodegenerative diseases, like Alzheimer’s, and Parkinson’s.
The complement system is a network of circulating and membrane-expressed proteins that recognize non-self-components. Its pathways help clear pathogens and apoptotic and dead cells by depositing non-terminal complement proteins on targeted cells to cause inflammation and mark apoptotic cells to be cleared by microglia. This removes their toxicity that can cause excitotoxicity and damage neighboring cells in RRMS. It can also aberrantly activate in SPMS and causes microglia to damage healthy CNS cells. Terminal complement components forming the membrane attack complex (MAC), or C5b-9 are tightly controlled to avoid harming healthy cell. It assembles terminal complement proteins (C5, C6, C7, C8, and C9) on a target membrane to create a pore that can cause cell lysis and death. It’s the final result of the complement cascade and a key innate immune system response. Most components can be produced in the CNS and increase in SPMS.
The microglia induced chronic pro-inflammatory environment also activates astrocytes.
Microglia can be activated by Th1 or Th17 T cells; microbial pathogens (PAMPs) recognized by TLRs or NLRs; intracellular components released by necrotic or apoptotic cells; fibrinogen; heat shock proteins, misfolded proteins (DAMPs), or components of the complement cascade. Microglia are activated early in MS and highly activated in SPMS.
Like other NDs, expanded and activated microglia is the primary mechanism behind astrogliosis. OS induced by inflammation and ROS/RNS injure astrocytes, causing them to change their cell morphology and molecular expressions in reactive astrogliosis. Astrocytes enter a reactive state and abnormally increase, turning OPCs to into reactive astrocytes instead of OLs. They make up most of the scar tissue, but sometimes interact with OPCs and fibromeningeal to create the scars surrounding demyelinated areas in MS. When activated they signal for more pro-inflammatory monocytes to enter the CNS, act as APCs by expressing MHC-II, secrete inflammatory cytokines, and stop supporting OLS and neurons. They also retract their endfeet from the glia limitans which affects BBB permeability and CSF flow in PVSs. They upregulate GFAP, vimentin, nestin, and synemin in this process. It stops tissue destruction spread, but the rigidity inhibits remyelination and axon regeneration. Reactive astrocytes also highly produce excessive ECM molecules, like CSPGs, proteoglycans, and glycosaminoglycan. These affect growth cone cytoskeleton and membrane components, inhibit axon growth, and prevent OPC maturation. They also oversecrete the FGF-2 protein that promotes OPC proliferation, but prevents them from maturing.
Ephrins (EPH) and their receptors are also secreted by astrocytes and increase in MS lesions. They induce axon growth cone collapse by activating axon-bound EPH tyrosine-receptor kinase. Astrocytes also directly affect CNS cell entry by regulating vascular adhesion-molecule-1 (VCAM-1), and intercellular adhesion-molecule-1 (ICAM-1) expressions that bind to lymphocyte receptors very late antigen-4 (VLA4), and lymphocyte function-associated antigen-1 (LFA-1). They also secrete chemokines like CCL-2 (MCP-1), CCL5 (RANTES), IP-10 (CXCL10), CXCL12 (SDF-1) and IL-8 (CXCL8), to attract B and T cells, monocytes, DCs, and microglia to lesion sites. Astrocytes also secrete GM-CSF, M-CSF, or TGF-β, which can regulate MHC Class II molecule expression by microglia and even their phagocytosis. This may be the main way they keep immune-mediated demyelination and neurodegeneration ongoing.
Astrocytes express the enzyme 4-galactosyltransferase 6 (B4GALT6) in MS lesions. It synthesizes signaling molecule lactosylceramide (LacCer), which highly increases expression in the CNS in SPMS. LacCer activates astrocytes in an autocrine manner, which induces GM-CSF and CCL2 genes. These activate microglia, and signal monocytes to enter the CNS. Activated microglia produce IL-1, IL-6, TN.F-α and ROS/RNS that cause damage in the CNS. Another feedback mechanism where activated immune cells can activate each other to promote more inflammation.
Astrocytes also express BAFF, that helps B-cells develop, survive, and produce immunoglobulins in normal physiological conditions in the CNS. This is upregulated in MS lesions, so astrocytes help drive B-cell-dependent autoimmunity too. B cells also form eLFs, that produce antibodies against myelin and non-myelin antigens. These damage axons and neurons by activating the complement cascade.
Astrocytes stimulated with IL-17 or IFN-γ induce nitric oxide synthase (iNOS).
IL-1 along with TGF-β and IFN-γ causes astrocytes to increase NO secretions, which is highly involved in neurodegeneration. NO also stimulates glutamate released from astrocytes to increase excitotoxicity depending on increased superoxide ion O2- production, which reacts with NO to form peroxynitrite (ONOO−) which causes neuron necrosis or apoptosis, depending on concentrations. ONOO− inactivates astrocyte glutamate transporters, directly damaging myelin, OLs, and axons. Decreased glutamate uptake by astrocyte transporters also adds to high extracellular glutamate, directly toxic to OLs, axons, and neurons. Their downregulated glutamate and potassium transporter expression on their cell surface leads to excitotoxicity. Excitotoxicity is from sustained glutamate receptor activation, which causes high influxes of Ca2+ into neurons, changing microtubules and NFL phosphorylation, that break down axon cytoskeletons. Glutamate excitotoxicity also decreases the threshold for complement activation on neuron surfaces. Complement positive microglia clusters in chronic WM lesions may be trying to remove damaged axons as they are just in chronic, but not acute lesions. Complement may activate to clear myelin debris or toxic products, but without clearance due to M1 microglia activation, they sustain the activated complement response. This leads to terminal complement production, C3a and C5a chemokine synthesis, and axon degeneration.
Fibrinogen also deposits at IRL rims and potently activates complement. A leaky BBB due to accumulated senescent endothelial cells with impaired tight junction structure may occur here too. Fibrinogen interacts with microglia via the CD11b/CD18 integrin receptor, activating perivascular microglia.
Astrocytes also help create an environment for remyelination. Impacts depend on timing after injury, lesion type, surrounding microenvironment, and interaction with other cell types and factors influencing their activation.
In healthy neural tissue, astrocytes have critical roles in providing energy, regulating blood flow, homeostasis of extracellular fluid, homeostasis of ions and transmitters, regulating synapse function, and remodeling synapses.
In parallel with the pathogenic role of both microglia and astrocytes in SPMS is the loss of their critical homeostatic functions that are needed for a healthy CNS.
There’s a fine balance between physiological and pathological functions in the CNS. In SPMS, with lower GSH, nitrosative and oxidative stress tips the balance toward pathalogical damage.
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