Deans' stroke musings: Lymphoid gene expression supports neuroprotective microglia function

Changing stroke rehab and research worldwide now.Time is Brain! trillions and trillions of neurons that DIE each day because there are NO effective hyperacute therapies besides tPA(only 12% effective). I have 523 posts on hyperacute therapy, enough for researchers to spend decades proving them out. These are my personal ideas and blog on stroke rehabilitation and stroke research. Do not attempt any of these without checking with your medical provider. Unless you join me in agitating, when you need these therapies they won't be there.

What this blog is for:

My blog is not to help survivors recover, it is to have the 10 million yearly stroke survivors light fires underneath their doctors, stroke hospitals and stroke researchers to get stroke solved. 100% recovery. The stroke medical world is completely failing at that goal, they don't even have it as a goal. Shortly after getting out of the hospital and getting NO information on the process or protocols of stroke rehabilitation and recovery I started searching on the internet and found that no other survivor received useful information. This is an attempt to cover all stroke rehabilitation information that should be readily available to survivors so they can talk with informed knowledge to their medical staff. It lays out what needs to be done to get stroke survivors closer to 100% recovery. It's quite disgusting that this information is not available from every stroke association and doctors group.

Thursday, November 27, 2025

Lymphoid gene expression supports neuroprotective microglia function

Now your competent? doctor and hospital can get human testing going.

Do you prefer your doctor, hospital and board of director's incompetence NOT KNOWING? OR NOT DOING?

Lymphoid gene expression supports neuroprotective microglia function

Nature (2025)Cite this article

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Abstract

Microglia, the innate immune cells of the brain, play a defining role in the progression of Alzheimer’s disease (AD)¹. The microglial response to amyloid plaques in AD can range from neuroprotective to neurotoxic². Here we show that the protective function of microglia is governed by the transcription factor PU.1, which becomes downregulated following microglial contact with plaques. Lowering PU.1 expression in microglia reduces the severity of amyloid disease pathology in mice and is linked to the expression of immunoregulatory lymphoid receptor proteins, particularly CD28, a surface receptor that is critical for T cell activation^3,4. Microglia-specific deficiency in CD28, which is expressed by a small subset of plaque-associated PU.1^low microglia, promotes a broad inflammatory microglial state that is associated with increased amyloid plaque load. Our findings indicate that PU.1^low CD28-expressing microglia may operate as suppressive microglia that mitigate the progression of AD by reducing the severity of neuroinflammation. This role of CD28 and potentially other lymphoid co-stimulatory and co-inhibitory receptor proteins in governing microglial responses in AD points to possible immunotherapy approaches for tr eating the disease by promoting protective microglial functions.

Main

Alzheimer’s disease is associated with diverse phenotypic changes in microglia^5,6, resulting in distinct microglial states, including protective (aimed at amyloid clearance and neuroprotection^7,8,9) and harmful (associated with microglia-driven neuroinflammation and toxicity)^10,11. These opposing states are accompanied by broad changes in gene expression^{12,13,14,15,16,17}, suggesting that defined transcriptional programs in microglia may govern their neuroprotective or neurotoxic functions. Here we present evidence that the neuroprotective state of microglia is governed by reduced expression of PU.1, a non-canonical pioneer transcription factor that functions as a master regulator of myeloid and lymphoid lineage differentiation^18,19,20. PU.1 regulates lineage-specific gene expression in a dose-dependent manner^21,22,23. More recently, PU.1 abundance in microglia has been linked to AD risk. Human genetic data show that a common variant, located within the 3′ untranslated region of the PU.1-encoding gene SPI1 and associated with reduced PU.1 expression in myeloid cells, correlates with delayed disease onset and reduced severity²⁴.

We found that PU.1 expression in microglia is regulated not only genetically but also epigenetically by the microenvironment. Using the amyloid-based 5xFAD mouse model of the disease²⁵, we identified a distinct subpopulation of microglia, among the previously described disease-associated microglia (DAM)¹² or neurodegenerative microglia¹³, that displays lower PU.1/Spi1 expression compared with the rest of the population (Fig. 1a, Supplementary Fig. 1a–h and Supplementary Table 1). These PU.1^low microglia preferentially co-localized with amyloid plaques, in both 5xFAD mice (Fig. 1b and Supplementary Fig. 1i) and individuals with AD (Fig. 1c and Supplementary Table 2), and increase in numbers with disease progression (Extended Data Fig. 1a). Unlike non-plaque-associated (distal microglia; Extended Data Fig. 1b) or PU.1^high plaque-associated microglia, this subpopulation appears independent of the activity of the essential microglial survival receptor CSF1R^26,27 (Fig. 1d,e and Extended Data Fig. 1b–d). The CSF1R-independent survival of PU.1^low microglia at plaques may reflect either their non-microglial origin or the engagement of alternative compensatory survival pathways. Lineage-tracing experiments using a microglia-specific genetic approach (translating ribosome affinity purification (TRAP) mice²⁸; Fig. 1d, Supplementary Fig. 2 and Supplementary Table 3) confirmed that the plaque-associated CSF1R-independent PU.1^low cells, despite showing low Csf1r gene expression (Supplementary Figs. 1h and 2f), are bona fide microglia.

**Fig. 1: Plaque-associated microglia include PU.1^low subpopulation.**

The PU.1^low microglial population at amyloid plaques may be established either through a pre-existing PU.1^low microglial subpopulation that is recruited specifically to plaques or by the downregulation of PU.1 expression in microglia in response to plaque-associated cues. Our data support the latter mechanism. Microglial accumulation at plaques and plaque-driven morphological and transcriptional responses depend on microglial surface receptor-mediated signalling^8,12,13,29, which involves activation of the tyrosine kinase (SYK)^9,30 and one of its key downstream targets (PLCγ2)^31,32. Microglia-specific inactivation of either SYK (5xFAD;Cx3cr1^CreERT2/+;Syk^fl/fl; referred to as 5xFAD;SYK-KO) or PLCγ2 (5xFAD;Cx3cr1^CreERT2/+;Plcg2^fl/fl; referred to as 5xFAD;PLCγ2-KO), which impairs microglial engagement with the plaques^9,30,32 (Fig. 1f), also significantly reduces the number of PU.1^low microglia (Fig. 1f and Extended Data Fig. 1e) and averts their CSF1R-independent survival at the plaques (Extended Data Fig. 1f).

Activation of SYK–PLCγ2-dependent signalling pathways in plaque-associated microglia occurs following the engagement of surface receptor proteins, including TREM2, CLEC7A and FcγR³³. To confirm that PU.1 downregulation is driven by surface receptor-mediated signalling, we exposed ex vivo isolated microglia to phosphatidylserine/phosphatidylcholine (PS/PC)-containing liposomes³¹ or β-1,6-glucan pustulan³⁴, which engage TREM2 and CLEC7A, respectively. Both ligands induced PU.1 downregulation (Extended Data Fig. 2a–d), which was attenuated by pharmacologic inhibition of downstream phospholipase C (PLC) activity³⁵ (Extended Data Fig. 2e,f). Conversely, activation of PLC³⁶ was sufficient to downregulate PU.1 even in the absence of external ligands (Extended Data Fig. 2e), confirming that activation of the SYK–PLCγ2 signalling pathways triggered by plaque-associated cues can regulate PU.1 expression in microglia.

The PU.1^low plaque-associated microglia displayed gene expression patterns that, in addition to promoting lipid metabolism and lysosomal function^37,38,39,40 (Extended Data Fig. 3a), showed a resemblance to lymphoid lineage cells (Fig. 2 and Extended Data Fig. 3). Microglial single-cell (Fig. 2a, Extended Data Fig. 3a,b and Supplementary Table 1), lineage-traced single-nucleus (Extended Data Fig. 3c–e and Supplementary Table 3) or spatial transcriptomics analyses (Fig. 2b,c, Extended Data Fig. 3f,g, Supplementary Fig. 3 and Supplementary Table 4) revealed that PU.1^low plaque-associated microglia express genes encoding immunoregulatory lymphoid receptors and signalling proteins, such as CD28, PD-1 (Pdcd1), PD-L1 (Cd274), CD5, CTLA-2A, CD52, LAT2 and CD72 (Extended Data Fig. 3d). The transcripts encoding these proteins are associated with ribosomes in 5xFAD microglia (Fig. 2d, Supplementary Fig. 4 and Supplementary Table 5), suggesting their translation and protein expression. Microglial lymphoid gene expression in PU.1^low disease-associated microglia (DAM) is not restricted to mice but is also observed in individuals with AD¹⁵ (Fig. 2e and Supplementary Fig. 5).