Weihua Zhao

Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis

The most common inherited form of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting adult motoneurons, is caused by dominant mutations in the ubiquitously expressed Cu2+/Zn2+ superoxide dismutase (SOD1). Recent studies suggest that glia may contribute to motoneuron injury in animal models of familial ALS. To determine whether the expression of mutant SOD1 (mSOD1G93A) in CNS microglia contributes to motoneuron injury, PU.1−/− mice that are unable to develop myeloid and lymphoid cells received bone marrow transplants resulting in donor-derived microglia. Donor-derived microglia from mice overexpressing mSOD1G93A, an animal model of familial ALS, transplanted into PU.1−/− mice could not induce weakness, motoneuron injury, or an ALS-like disease. To determine whether expression of mSOD1G93A in motoneurons and astroglia, as well as microglia, was required to produce motoneuron disease, PU.1−/− mice were bred with mSOD1G93A mice. In mSOD1G93A/PU.1−/− mice, wild-type donor-derived microglia slowed motoneuron loss and prolonged disease duration and survival when compared with mice receiving mSOD1G93A expressing cells or mSOD1G93A mice. In vitro studies confirmed that wild-type microglia were less neurotoxic than similarly cultured mSOD1G93A microglia. Compared with wild-type microglia, mSOD1G93A microglia produced and released more superoxide and nitrite+nitrate, and induced more neuronal death. These data demonstrate that the expression of mSOD1G93A results in activated and neurotoxic microglia, and suggests that the lack of mSOD1G93A expression in microglia may contribute to motoneuron protection. This study confirms the importance of microglia as a double-edged sword, and focuses on the importance of targeting microglia to minimize cytotoxicity and maximize neuroprotection in neurodegenerative diseases.

CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS.

Neuroinflammation, marked by gliosis and infiltrating T cells, is a prominent pathological feature in diverse models of dominantly inherited neurodegenerative diseases. Recent evidence derived from transgenic mice ubiquitously overexpressing mutant Cu2+/Zn2+ superoxide dismutase (mSOD1), a chronic neurodegenerative model of inherited amyotrophic lateral sclerosis (ALS), indicates that glia with either a lack of or reduction in mSOD1 expression enhance motoneuron protection and slow disease progression. However, the contribution of T cells that are present at sites of motoneuron injury in mSOD1 transgenic mice is not known. Here we show that when mSOD1 mice were bred with mice lacking functional T cells or CD4+ T cells, motoneuron disease was accelerated, accompanied by unexpected attenuated morphological markers of gliosis, increased mRNA levels for proinflammatory cytokines and NOX2, and decreased levels of trophic factors and glial glutamate transporters. Bone marrow transplants reconstituted mice with T cells, prolonged survival, suppressed cytotoxicity, and restored glial activation. These results demonstrate for the first time in a model of chronic neurodegeneration that morphological activation of microglia and astroglia does not predict glial function, and that the presence of CD4+ T cells provides supportive neuroprotection by modulating the trophic/cytotoxic balance of glia. These glial/T-cell interactions establish a novel target for therapeutic intervention in ALS and possibly other neurodegenerative diseases.

Microglia in ALS: The Good, The Bad, and The Resting

Inflammation, including microglial activation and T cell infiltration, is a neuropathological hallmark of amyotrophic lateral sclerosis (ALS), a rapidly progressing neurodegenerative disease. The identification of mutations in the gene for Cu2+/Zn2+ superoxide dismutase (SOD1) from patients with an inherited form of ALS enabled the creation of transgenic mice overexpressing mutant forms of SOD1 (mSOD1) which develop a motoneuron disease that resembles the disease seen in ALS patients. These transgenic mice display similar inflammatory reactions at sites of motoneuron injury as detected in ALS patients, enabling the observation that this inflammation is not simply a late consequence of motoneuron degeneration, but actively contributes to the balance between neuroprotection and neurotoxicity. The microglial and T cell activation states influence the rate of disease progression. Initially, microglia and T cells can slow disease progression, while they may later contribute to the acceleration of disease. Accumulation of intracellular and extracellular misfolded mSOD1 may be key events regulating the transformation from neuroprotective alternatively activated M2 microglia to cytotoxic classically activated M1 microglia. Intracellular and extracellular mSOD1 utilizing different pathways may enhance the production and release of reactive oxygen species (ROS) and augment the inflammatory cytokine cascade from microglia. These ROS and cytokines may increase the susceptibility of motoneurons to glutamate toxicity and inhibit the function and expression of astrocytic glutamate transporters resulting in further neurotoxicity. Thus, the cumulative evidence suggests that inflammation plays a central role in ALS and manipulating these microglial effector functions may potentially modify the outcome of this devastating disease.

Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS

Neuroinflammation is a prominent pathological feature in the spinal cords of patients with amyotrophic lateral sclerosis (ALS), as well as in transgenic mouse models of inherited ALS, and is characterized by activated microglia. Earlier studies showed that activated microglia play important roles in both motoneuron protection and injury. More recent studies investigating the pathoprogression of disease in ALS mice have demonstrated that the in vivo activation states of microglia, including their anti- versus pro-inflammatory responses, are best characterized as a continuum between two extreme activation states which are represented as a neuroprotective M2 (alternatively-activated) phenotypic state or an injurious/toxic M1 (classically-activated) state; a more complete understanding and determination the temporal transformation of microglia activation states in the ALS disease pathoprogression is therefore warranted. In the current study, we demonstrated a phenotypic and functional transformation of adult ALS mice microglia that overexpress mutant superoxide dismutase (mSOD1). mSOD1 microglia isolated from ALS mice at disease onset expressed higher levels of Ym1, CD163 and BDNF (markers of M2) mRNA and lower levels of Nox2 (a marker of M1) mRNA compared with mSOD1 microglia isolated from ALS mice at end-stage disease. More importantly, when co-cultured with motoneurons, these mSOD1 M2 microglia were neuroprotective and enhanced motoneuron survival than similarly co-cultured mSOD1 M1 microglia; end-stage mSOD1 M1 microglia were toxic to motoneurons. Our study documents that adult microglia isolated from ALS mice at disease onset have an M2 phenotype and protect motoneurons whereas microglia isolated from end-stage disease ALS mice have adopted an M1 phenotype and are neurotoxic supporting the dual phenotypes of microglia and their transformation during disease pathoprogression in these mice. Thus, harnessing the neuroprotective potential of microglia may provide novel avenues for ALS therapies.

Endogenous regulatory T lymphocytes ameliorate amyotrophic lateral sclerosis in mice and correlate with disease progression in patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a relentless and devastating adult-onset neurodegenerative disease with no known cure. In mice with amyotrophic lateral sclerosis, CD4+ T lymphocytes and wild-type microglia potentiate protective inflammatory responses and play a principal role in disease pathoprogression. Using this model, we demonstrate that endogenous T lymphocytes, and more specifically regulatory T lymphocytes, are increased at early slowly progressing stages, augmenting interleukin-4 expression and protective M2 microglia, and are decreased when the disease rapidly accelerates, possibly through the loss of FoxP3 expression in the regulatory T lymphocytes. Without ex vivo activation, the passive transfer of wild-type CD4+ T lymphocytes into amyotrophic lateral sclerosis mice lacking functional T lymphocytes lengthened disease duration and prolonged survival. The passive transfer of endogenous regulatory T lymphocytes from early disease stage mutant Cu2+/Zn2+ superoxide dismutase mice into these amyotrophic lateral sclerosis mice, again without ex vivo activation, were substantially more immunotherapeutic sustaining interleukin-4 levels and M2 microglia, and resulting in lengthened disease duration and prolonged survival; the stable disease phase was extended by 88% using mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes. A potential mechanism for this enhanced life expectancy may be mediated by the augmented secretion of interleukin-4 from mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes that directly suppressed the toxic properties of microglia; flow cytometric analyses determined that CD4+/CD25+/FoxP3+ T lymphocytes co-expressed interleukin-4 in the same cell. These observations were extended into the amyotrophic lateral sclerosis patient population where patients with more rapidly progressing disease had decreased numbers of regulatory T lymphocytes; the numbers of regulatory T lymphocytes were inversely correlated with disease progression rates. These data suggest a cellular mechanism whereby endogenous regulatory T lymphocytes are immunocompetent and actively contribute to neuroprotection through their interactions with microglia. Furthermore, these data suggest that immunotherapeutic interventions must begin early in the pathogenic process since immune dysfunction occurs at later stages. Thus, the cumulative mouse and human amyotrophic lateral sclerosis data suggest that increasing the levels of regulatory T lymphocytes in patients with amyotrophic lateral sclerosis at early stages in the disease process may be of therapeutic value, and slow the rate of disease progression and stabilize patients for longer periods of time.

Extracellular mutant SOD1 induces microglial-mediated motoneuron injury

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase‐1 (mSOD1) cause the non‐cell‐autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1‐activated microglia. To dissociate these potential mSOD1‐mediated neurotoxic mechanisms, the effects of extracellular human mSOD1G93A or mSOD1G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1G93A or mSOD1G85R did induce the morphological and functional activation of microglia, increasing their release of pro‐inflammatory cytokines and free radicals. Furthermore, only when microglia was co‐cultured with motoneurons did extracellular mSOD1G93A injure motoneurons. The microglial activation mediated by mSOD1G93A was attenuated using toll‐like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1G93A is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS.

Immune-mediated mechanisms in the pathoprogression of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with selective loss of upper and lower motor neurons. At sites of motor neuron injury, neuroinflammation is a prominent pathological finding and is characterized by microglial activation, astrogliosis, and infiltration of monocytes and T-cells. Both innate and adaptive immune responses actively influence disease progression in animal models and in ALS patients, and promote neuroprotection or neurotoxicity at different stages of disease. The early immune reaction to signals from injured motor neurons is to rescue and repair damaged tissue. As disease accelerates, a shift occurs from beneficial immune responses (involving M2 microglia and regulatory T-cells) to deleterious immune responses (involving M1 microglia and Th1 cells). In this review, we underscore the importance of immune-mediated mechanisms in the pathogenesis of ALS and discuss the alterations and distinct phenotypes of immune cells at the different stages of disease. The better we understand the dynamic changes that occur within the immune system over the course of disease, the better we will be able to develop effective therapeutic regimens in ALS.

Regulatory T‐lymphocytes mediate amyotrophic lateral sclerosis progression and survival

In amyotrophic lateral sclerosis (ALS) mice, regulatory T‐lymphocytes (Tregs) are neuroprotective, slowing disease progression. To address whether Tregs and FoxP3, a transcription factor required for Treg function, similarly influence progression rates of ALS patients, T‐lymphocytes from patients were assessed by flow cytometry. Both numbers of Tregs and their FoxP3 protein expressions were reduced in rapidly progressing ALS patients and inversely correlated with progression rates. The mRNA levels of FoxP3, TGF‐β, IL4 and Gata3, a Th2 transcription factor, were reduced in rapidly progressing patients and inversely correlated with progression rates. Both FoxP3 and Gata3 were accurate indicators of progression rates. No differences in IL10, Tbx21, a Th1 transcription factor or IFN‐γ expression were found between slow and rapidly progressing patients. A 3.5‐year prospective study with a second larger cohort revealed that early reduced FoxP3 levels were indicative of progression rates at collection and predictive of future rapid progression and attenuated survival. Collectively, these data suggest that Tregs and Th2 lymphocytes influence disease progression rates. Importantly, early reduced FoxP3 levels could be used to identify rapidly progressing patients.

Mutant SOD1G93A microglia are more neurotoxic relative to wild-type microglia

Recent studies suggest that microglia over‐expressing mutant human superoxide dismutase (mSOD1G93A) may contribute to motoneuron death in a transgenic mouse model of familial amyotrophic lateral sclerosis. To further assess the relative neurotoxicity of wild‐type microglia, mSOD1G93A microglia, and microglia over‐expressing wild‐type human SOD1, we used primary cultures of microglia and motoneurons in the presence and absence of lipopolysaccharide stimulation. Following activation with lipopolysaccharide, mSOD1G93A microglia released more nitric oxide, more superoxide, and less insulin‐like growth factor‐1 than wild‐type microglia. In microglia/motoneuron co‐cultures, mSOD1G93A microglia induced more motoneuron death and decreased neurite numbers and length compared with wild‐type microglia. Mutant SOD1G93A microglia also induced more motoneuron injury than microglia over‐expressing wild‐type human SOD1 in microglia/motoneuron co‐cultures. Motoneuron survival was inversely correlated with nitrateu2003+u2003nitrite concentrations in mSOD1G93A co‐cultures, suggesting the important role of nitric oxide in microglia‐induced motoneuron injury. Thus, relative to wild‐type microglia, mSOD1G93A microglia were more neurotoxic and induced more motoneuron injury than similarly treated wild‐type microglia.

Neuroinflammation modulates distinct regional and temporal clinical responses in ALS mice

An inflammatory response is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a relentless and devastating degenerative disease of motoneurons. This response is not simply a late consequence of motoneuron degeneration, but actively contributes to the balance between neuroprotection and neurotoxicity; initially infiltrating lymphocytes and microglia slow disease progression, while later, they contribute to the acceleration of disease. Since motor weakness begins in the hindlimbs of ALS mice and only later involves the forelimbs, we determined whether differential protective versus injurious inflammatory responses in the cervical and lumbar spinal cords explained the temporally distinct clinical disease courses between the limbs of these mice. Densitometric evaluation of immunohistochemical sections and quantitative RT-PCR (qRT-PCR) demonstrated that CD68 and CD11c were differentially increased in their spinals cords. qRT-PCR revealed that protective and anti-inflammatory factors, including BDNF, GDNF, and IL-4, were increased in the cervical region compared with the lumbar region. In contrast, the toxic markers TNF-α, IL-1β and NOX2 were not different between ALS mice cervical and lumbar regions. T lymphocytes were observed infiltrating lumbar spinal cords of ALS mice prior to the cervical region; mRNA levels of the transcription factor gata-3 (Th2 response) were differentially elevated in the cervical cord of ALS mice whereas t-bet (Th1 response) was increased in the lumbar cord. These results reinforce the important balance between specific protective/injurious inflammatory immune responses in modulating clinical outcomes and suggest that the delayed forelimb motor weakness in ALS mice is partially explained by augmented protective responses in the cervical spinal cords.