Chronically activated microglia in ALS gradually lose their immune functions and develop unconventional proteome.

Neuroinflammation and chronic activation of microglial cells are the prominent features of amyotrophic lateral sclerosis (ALS) pathology. While alterations in the mRNA profile of diseased microglia have been well documented, the actual microglia proteome remains poorly characterized. Here we performed a functional characterization together with proteome analyses of microglial cells at different stages of disease in the SOD1-G93A model of ALS. Functional analyses of microglia derived from the lumbar spinal cord of symptomatic mice revealed: (i) remarkably high mitotic index (close to 100% cells are Ki67+) (ii) significant decrease in phagocytic capacity when compared to age-matched control microglia, and (iii) diminished response to innate immune challenges in vitro and in vivo. Proteome analysis revealed a development of two distinct molecular signatures at early and advanced stages of disease. While at early stages of disease, we identified several proteins implicated in microglia immune functions such as GPNMB, HMBOX1, at advanced stages of disease microglia signature at protein level was characterized with a robust upregulation of several unconventional proteins including rootletin, major vaults proteins and STK38. Upregulation of GPNMB and rootletin has been also found in the spinal cord samples of sporadic ALS. Remarkably, the top biological functions of microglia, in particular in the advanced disease, were not related to immunity/immune response, but were highly enriched in terms linked to RNA metabolism. Together, our results suggest that, over the course of disease, chronically activated microglia develop unconventional protein signatures and gradually lose their immune identity ultimately turning into functionally inefficient immune cells.

Optimization of a lipid nanoparticle-based protocol for RNA transfection into primary mononuclear phagocytes.

The effective delivery of synthetic RNA into mononuclear phagocytes is a prerequisite for experimental research and therapeutic development. However, traditional methods are highly ineffective and toxic for these cells. Here, we aimed to optimize a transfection protocol for primary bone marrow-derived phagocytes, specifically dendritic cells and macrophages, using lipid nanoparticles generated by microfluidics. Our results show that a lipid mixture similar to that used in Moderna's COVID-19 messenger RNA vaccine outperforms the others tested. Improved messenger RNA transfection can be achieved by replacing uridine with methylpseudouridine but not methoxyuridine, which interferes with transfection. The addition of diphenyleneiodonium or apocynin can enhance transfection in a cell type-dependent manner without adverse effects, while apolipoprotein E provides no added value. These optimized transfection conditions can also be used for microRNA agonists and antagonists. In sum, this study offers a straightforward, highly efficient, reproducible, and nontoxic protocol to deliver RNA into different primary mononuclear phagocytes in culture.

Microglia-derived galectin-3 in neuroinflammation; a bittersweet ligand?

Galectins are soluble ß-galactoside-binding proteins found in all multicellular organisms. Galectins may act as danger-associated molecular patterns in innate immunity and/or as pattern-recognition receptors that bind to pathogen-associated molecular patterns. Among different galectin family members, galectin-3 has been the focus of studies in neurodegenerative diseases in recent years. This lectin modulates brain innate immune responses, microglia activation patterns in physiological and pathophysiological settings in a context-dependent manner. Galectin-3 is considered as a pivotal tuner of macrophage and microglial activity. Indeed galectin-3 acts as a double edged sword in neuroinflammatory context and this multimodal lectin has diverse roles in physiological and pathophysiological conditions. Better understanding of galectin-3 physiology (its extracellular and intracellular actions) and structure (its C terminus vs. N terminus) is instrumental to design molecules that selectively modulate galectin-3 function toward neuroprotective phenotypes. Several experimental studies using different approaches and methods have demonstrated both protective and deleterious effects of galectin-3 in neuroinflammatory diseases. According to the crucial role of galectin-3 in modulation of innate immune response in brain, it is an attractive target in drug discovery of neurodegenerative diseases. The current insight attempts to provide an updated and balanced discussion on the role of galectin-3 as a complex endogenous immune modulator. This helps to have a better insight into the development of galectin-3 modulators with translational value in different neurological disorders including stroke and neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

Immunity in amyotrophic lateral sclerosis: blurred lines between excessive inflammation and inefficient immune responses.

Despite wide genetic, environmental and clinical heterogeneity in amyotrophic lateral sclerosis, a rapidly fatal neurodegenerative disease targeting motoneurons, neuroinflammation is a common finding. It is marked by local glial activation, T cell infiltration and systemic immune system activation. The immune system has a prominent role in the pathogenesis of various chronic diseases, hence some of them, including some types of cancer, are successfully targeted by immunotherapeutic approaches. However, various anti-inflammatory or immunosuppressive therapies in amyotrophic lateral sclerosis have failed. This prompted increased scrutiny over the immune-mediated processes underlying amyotrophic lateral sclerosis. Perhaps the biggest conundrum is that amyotrophic lateral sclerosis pathogenesis exhibits features of three otherwise distinct immune dysfunctions-excessive inflammation, autoimmunity and inefficient immune responses. Epidemiological and genome-wide association studies show only minimal overlap between amyotrophic lateral sclerosis and autoimmune diseases, so excessive inflammation is usually thought to be secondary to protein aggregation, mitochondrial damage or other stresses. In contrast, several recently characterized amyotrophic lateral sclerosis-linked mutations, including those in TBK1, OPTN, CYLD and C9orf72, could lead to inefficient immune responses and/or damage pile-up, suggesting that an innate immunodeficiency may also be a trigger and/or modifier of this disease. In such cases, non-selective immunosuppression would further restrict neuroprotective immune responses. Here we discuss multiple layers of immune-mediated neuroprotection and neurotoxicity in amyotrophic lateral sclerosis. Particular focus is placed on individual patient mutations that directly or indirectly affect the immune system, and the mechanisms by which these mutations influence disease progression. The topic of immunity in amyotrophic lateral sclerosis is timely and relevant, because it is one of the few common and potentially malleable denominators in this heterogenous disease. Importantly, amyotrophic lateral sclerosis progression has recently been intricately linked to patient T cell and monocyte profiles, as well as polymorphisms in cytokine and chemokine receptors. For this reason, precise patient stratification based on immunophenotyping will be crucial for efficient therapies.

Optineurin Insufficiency Disbalances Proinflammatory and Anti-inflammatory Factors by Reducing Microglial IFN-ß Responses.

Mutations in a ubiquitin (Ub)-binding adaptor protein optineurin have been found in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease with a prominent neuroinflammatory component. Unlike more frequent ALS mutations which cause disease by gaining toxic properties such as aggregation, mutated optineurin is thought to cause disease by loss-of-function, highlighting its neuroprotective role. Optineurin regulates inflammatory signaling by acting as a scaffold for Tank-binding kinase 1 (TBK1) activation and interferon (IFN)-ß production in peripheral immune cells. The relevance of this pathway in the CNS is unclear. To investigate IFN-ß pathway as a potential mechanism of optineurin-mediated protection from neurodegeneration, we have generated a mouse model in which the Ub-binding region of optineurin was deleted (Optn470T), mimicking C-terminal truncations found in patients. Here we report reduced TBK1 activation and IFN-ß production in primary microglia from Optn470T model upon Toll-like receptor (TLR) stimulation. Likewise, we found diminished expression and activation of several transcription factors that support the amplification loop for IFN-ß production including STAT1, IRF7 and IRF9. Notably, although optineurin was also reported to block proinflammatory transcription factor NF-κB, normal NF-κB activation and TNF production were found in Optn470T microglia. However, expression of both proinflammatory and anti-inflammatory factors distal to IFN-ß was diminished, and could be restored upon IFN-ß supplementation. Taken together with the recent discoveries of TBK1 mutations as an important genetic factor in ALS, our results open up the possibility that disruption of optineurin/TBK1-mediated IFN-ß axis leads to an immune failure in containing neuronal damage, which could predispose to neurodegeneration.

Galectin-3: mediator of microglia responses in injured brain.

Galectin-3 is a pleiotropic protein involved in cell activation, proliferation and migration and plays a pivotal part as an inflammatory mediator in neurodegeneration. Galectin-3 is associated with microglial activation and proliferation after ischemia. Given its putative role as a dynamic fine-tuner of microglia, activation of Galectin-3 provides molecular cues in design of new immunomodulatory strategies for stroke management. This review summarizes recent evidence on the role of Galectin-3 as a mediator of immune responses in damaged brain and mechanisms employed by Galectin-3 to affect microglial function.

Diverging mRNA and Protein Networks in Activated Microglia Reveal SRSF3 Suppresses Translation of Highly Upregulated Innate Immune Transcripts.

Uncontrolled microglial activation may lead to the development of inflammation-induced brain damage. Here, we uncover a ribosome-based mechanism/checkpoint involved in control of the innate immune response and microglial activation. Using an in vivo model system for analysis of the dynamic translational state of microglial ribosomes, with mRNAs as input and newly synthesized peptides as an output, we find a marked dissociation of microglia mRNA and protein networks following innate immune challenge. Highly upregulated and ribosome-associated mRNAs were not translated, resulting in two distinct microglial molecular signatures, a highly specialized pro-inflammatory mRNA signature and an immunomodulatory/homeostatic protein signature. We find that this is due to specific translational suppression of highly expressed mRNAs through a 3' UTR-mediated mechanism involving the RNA-binding protein SRSF3. This discovery suggests avenues for therapeutic modulation of innate immune response in resident microglia.

IL-10 Controls Early Microglial Phenotypes and Disease Onset in ALS Caused by Misfolded Superoxide Dismutase 1.

While reactive microgliosis is a hallmark of advanced stages of amyotrophic lateral sclerosis (ALS), the role of microglial cells in events initiating and/or precipitating disease onset is largely unknown. Here we provide novel in vivo evidence of a distinct adaptive shift in functional microglial phenotypes in preclinical stages of superoxide dismutase 1 (SOD1)-mutant-mediated disease. Using a mouse model for live imaging of microglial activation crossed with SOD1(G93A) and SOD1(G37R) mouse models, we discovered that the preonset phase of SOD1-mediated disease is characterized by development of distinct anti-inflammatory profile and attenuated innate immune/TLR2 responses to lipopolysaccharide (LPS) challenge. This microglial phenotype was associated with a 16-fold overexpression of anti-inflammatory cytokine IL-10 in baseline conditions followed by a 4.5-fold increase following LPS challenge. While infusion of IL-10R blocking antibody, initiated at day 60, caused a significant increase in markers of microglial activation and precipitated clinical onset of disease, a targeted overexpression of IL-10 in microglial cells, delivered via viral vectors expressed under CD11b promoter, significantly delayed disease onset and increased survival of SOD1(G93A) mice. We propose that the high IL-10 levels in resident microglia in early ALS represent a homeostatic and compensatory "adaptive immune escape" mechanism acting as a nonneuronal determinant of clinical onset of disease. Significance statement: We report here for the first time that changing the immune profile of brain microglia may significantly affect clinical onset and duration of disease in ALS models. We discovered that in presymptomatic disease microglial cells overexpress anti-inflammatory cytokine IL-10. Given that IL-10 is major homeostatic cytokine and its production becomes deregulated with aging, this may suggest that the capacity of microglia to adequately produce IL-10 may be compromised in ALS. We show that blocking IL-10 increased inflammation and precipitated clinical disease onset, whereas overexpression of IL-10 in microglia using a gene therapy approach significantly delayed disease onset and increased survival of ALS mice. Based on our results, we propose that targeted overexpression of IL-10 in microglia may have therapeutic potential in ALS.