Biology Direct

Microglial activation is a central component of neuroinflammatory responses in many brain pathologies. Increasing evidence indicates that microglial phenotype is tightly linked to cellular metabolism, with pro-inflammatory activation associated with enhanced glycolytic flux. Lactate, traditionally considered a metabolic substrate, has recently emerged as a signaling molecule capable of modulating immune responses. However, its direct impact on microglial inflammatory activation remains incompletely understood. In the present study, we investigated the effects of lactate on microglial phenotype under inflammatory conditions using primary rat microglial cultures stimulated with lipopolysaccharide (LPS). Microglial activation was assessed through the expression of phenotypic markers, cytokine production, and secreted chemokine profiles. LPS stimulation induced a strong pro-inflammatory response characterized by increased CD86 expression, elevated TNF-alpha secretion, and enhanced release of several pro-inflammatory chemokines. Post-treatment with sodium L-lactate significantly attenuated these inflammatory responses, reducing pro-inflammatory marker expression and cytokine secretion, while restoring the anti-inflammatory marker CD206. To explore the relevance of these findings in a pathological context, the effects of lactate were further examined in a neonatal rat model of hypoxia-ischemia. Sodium L-lactate administration after injury reduced microglial activation and promoted a shift toward an anti-inflammatory phenotype in cortical regions, whereas hippocampal microglia showed a more limited response. Together, these results demonstrate that lactate directly modulates microglial inflammatory activation and cytokine production in vitro and suggest that lactate-mediated metabolic signaling may contribute in vivo to the regulation of neuroinflammatory responses.

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.

Translation initiation is a critical stage of protein synthesis in prokaryotes, where ribosomes assemble on mRNA to begin decoding genetic information. The interactions between ribosomal RNA (rRNA) and mRNA significantly influence this process, with features like ribosomal binding site (RBS) affecting ribosomal dynamics. However, direct experimental validation of these types of interaction is yet to be provided. In this study, we developed an optimized small sub-unit sequencing (SSU-seq) protocol for Escherichia coli, enabling the detailed examination of ribosomal complexes and subunits dynamics. Unlike Ribo-seq, which primarily maps fully assembled ribosomes, SSU-seq captures both ribosomal subunits and intact ribosomes, offering a comprehensive view of translation initiation and elongation events. By integrating experimental data with predictive models, our study advances the understanding of translation regulation in prokaryotes and underscores the utility of SSU-seq in decoding the complexities of ribosome-mRNA interactions. Our analysis reveals distinct patterns of small subunit distribution across the entire mRNAs with higher density near positions with stronger rRNA-mRNA hybridization. The results may suggest that rRNA-mRNA hybridization positions along the entire transcript facilitate efficient start codon recognition by maintaining proximity between the small subunit and the mRNA.

During phage infection, phages reprogram their host to secure energy, building blocks for their genome, and proteins, as well as the transcription and translation machinery required to replicate their genome and produce phage particles. This also includes reprogramming the host transcriptome to modify host gene expression and align it with the needs of the phage during infection. In this study, we present a putative early-expressed protein, Gp204 of Mosigvirus AV110, renamed Host transcription modulator (Htm), that has a broad impact on the extended-spectrum beta-lactamase (ESBL)- associated E. coli transcriptome. Overexpression of Htm decreased the growth rate of ESBL-E. coli and caused DNA condensation. Htm is structurally similar to bacterial enhancer-binding proteins, binds ssDNA, and alters the expression levels of over 700 E. coli genes when expressed. The majority of these genes were upregulated and are involved in multiple pathways, such as galactose metabolism and pentose and glucuronate interconversions, which may promote the synthesis of purines, pyrimidines, and histidine, all of which are required for the replication, transcription, and translation of the phage genome. Furthermore, energy production is likely to be upregulated by increased availability of acetyl-CoA, resulting from, among other factors, fatty acid degradation. Htm also downregulated the expression of the structural genes involved in flagellar assembly and multiple two-component systems. In conclusion, Htm could promote phage progeny production by increasing the availability of nucleotides, amino acids, and energy, while inhibiting non-essential processes, such as flagella formation.

Forms I&prime, I&Prime, and I RuBisCOs were recently identified in bacterial metagenome-assembled genomes (MAGs) and are significant for evolutionarily bridging form I, which is responsible for the major part of carbon fixation on Earth, and other forms. Pioneering studies focused mainly on the biochemical and structural characteristics of "form I-related" RuBisCOs and deduced the evolution of this protein family over phylogenetic relationships among form I and form I-related sequences. We here surveyed form I-related sequences in public databases and conducted phylogenetic analyses incorporating the updated diversity of form I-related RuBisCOs. This study yielded two major outcomes regarding RuBisCO evolution. First, the analyses presented here suggest uncertainties overlooked in the previously published studies. It appeared to be difficult to conclude (i) the phylogenetic relationship among forms I, I&prime, and I&Prime, (ii) the monophyly of form I&prime, or (iii) the monophyly of form I. Second, we identified a gene encoding form I RuBisCO in an archaeal MAG, and this protein is likely involved in the pentose bisphosphate pathway. The results presented here suggest that the evolutionary route leading to form I in RuBisCO evolution remains unsettled.

Histomonosis, caused by Histomonas meleagridis, is a severe disease in poultry with main pathological lesions in ceca and liver, leading to significant animal suffering and economic losses, with no licensed treatments available. While cecal pathology and microbiota disruption have been documented, the metabolic consequences of infection and vaccination remain poorly understood. Here, we used untargeted LC-MS metabolomics to profile cecal tissue and luminal digesta in a controlled vaccination-challenge chicken trial (n=100, 25 birds/group), including uninfected and unvaccinated but challenged controls, together with birds vaccinated either cloacally, or orally via an edible gel following challenge, across five post-challenge timepoints. Results showed that H. meleagridis infection caused extensive, time-dependent disruption of the cecal metabolome, especially at 7-14 days post-infection (DPI). At DPI 14, 325 tissue and 359 digesta metabolites were significantly altered in unvaccinated challenged birds. Infection was associated with depletion of microbiota-related metabolites, including dicarboxylic acids, tryptophan indoles, phytochemical derivatives, bilirubin catabolites, phenolics, vitamins, and secondary bile acids, alongside accumulation of microbial substrates and fermentation products. Changes in the host-associated metabolites indicated mucosal inflammation, epithelial injury, mitochondrial and amino acid metabolic disturbance, oxidative stress, and membrane lipid remodeling. Cloacal vaccination provided greater metabolic protection than oral vaccination, with 78.3% of disrupted metabolites preserved at DPI 14, compared with 60.7% vaccination by oral gel delivery, pointing towards the importance of vaccine take. By DPI 21, cloacally vaccinated birds showed nearly complete metabolic recovery across most pathway groups, whereas gel-vaccinated birds retained broader metabolic disturbance. These findings reveal histomonosis as a disruption of the cecal host-microbiota metabolic interface and provide a metabolite-level framework for future studies of pathogenesis, vaccine development, and prevention strategies.

CRISPR-Cas is an adaptive immunity system that protects prokaryotes from viruses and other genetic parasites. CRISPR-Cas immunity is guided by short DNA sequences, called spacers, that are acquired upon encounters with parasites and stored in the CRISPR array. Although the mechanisms driving spacer acquisition are relatively well understood, the timespan of CRISPR immune memory and, consequently, the very nature of CRISPR-Cas as a short-term or long-term defense mechanism remain controversial. Because CRISPR arrays have limited size, CRISPR-Cas systems face a fundamental trade-off between acquiring new spacers against recent infections and retaining old spacers for long-term defense. To investigate how CRISPR-Cas systems resolve this "fast response-versus-memory" trade-off, we developed a stochastic model that captures CRISPR array dynamics under varying abundances of endemic and episodic viruses. Simulations reveal three distinct evolutionary regimes that result in short-term, long-term, and "dual memory" arrays. These regimes are governed by the ratio of two measurable parameters: the length of the CRISPR array and the time required for transient epidemics to decay below basal endemic viral abundance. Spontaneous division of labor emerges in the dual memory regime, with spacers near the leader and distal ends specialized in short-term and long-term memory, respectively. Within-array division of labor results in a U-shaped longitudinal profile for the probability of finding the targets of CRISPR immunity among the local virome. Such profiles, that have been empirically observed in CRISPR arrays from the human gut microbiome, could inform future research on the environmental persistence of poorly characterized viromes.

Phytoplankton sustain all marine ecosystems and are key components of the carbon cycle being responsible for ~50% of the global primary production. A major hypothesis to explain their coexistence with highly efficient lytic viruses is antiviral resistance. Although intracellular and extracellular mechanisms have been shown to underly such resistance and while its determinism is commonly associated to a genomic polymorphism or a (random or virus induced) phenotypic plasticity, comparisons of the impact of those molecular determinants on phytoplankton-virus population dynamic are genuinely lacking. We combined 12 epidemiological models with an unprecedented set of parameter estimates derived from experimental studies of the ubiquitous species Ostreococcus tauri and its prasinoviruses to provide such comprehensive predictions. We show that 'bet-hedging' strategies corresponding to random phenotypic switches between susceptible and resistant cells provide the strongest barrier to virus emergence. When such resistance is associated to intracellular mechanisms the virus' R0 is at least halved because of the strong 'dilution effect' produced by the 'dead-end' hosts. In addition, once the virus has spread, both random and virus-induced phenotypic plasticity strongly stabilize the otherwise 'boom and bust' phytoplankton-virus dynamics, which is highly consistent with the experimental dynamics observed for such interactions.

A significant fraction of archaeal genomes have their genomic content distributed across two or more DNA molecules, yet the characteristics and evolution of these different replicons remain poorly understood. Here, we investigated the genomic and functional organization of secondary replicons in Halobacteriota using 211 complete genomes, comprising 766 replicons, including 211 primary chromosomes (chr1), and 555 secondary replicons. Comparative analyses of genomic features, including replicon size, GC content, and codon usage, revealed a continuous distribution of secondary replicons from plasmid-like to chromosome-like characteristics. Larger secondary replicons showed progressively more chromosome-like genomic and functional properties, including greater protein-family overlap with chr1 and a higher proportion of conserved gene families. At the phylum level, functional profiles substantially overlapped among replicon types, providing further support for a continuum rather than discrete functional classes. Larger replicons also showed increased protein-family duplication, while pangenome analysis revealed a greater proportion of conserved gene families, with accessory families most abundant at intermediate sizes and rare and singleton families declining with increasing replicon size. Together, these results support a model in which secondary replicons in Halobacteriota span a genomic and functional continuum from plasmid-like to chromosome-like states , with increasing size associated with greater conservation, gene duplication and progressively more chromosome-like gene content and functional profile.

We have examined how ibogaine may enhance synaptic plasticity and neural repair. Extracellular efflux analysis showed that ibogaine (3, 10 or 30 M) produced a rapid (<5 minutes) and concentration-dependent suppression of mitochondrial function and glycolysis in mouse cortical neurons. Cell-based imaging demonstrated this metabolic inhibition was preceded by an elevation of the mitochondrial membrane potential and followed by increased mitochondrial reactive oxygen species levels. Like ibogaine, which blocks potassium channels, mitochondrial Kv1.3 channel (mitoKv1.3) inhibitors produce concordant changes over a similar timeframe. Following treatment with ibogaine (3, 10 or 30 M) for 4 hours, a marked concentration-dependent rebound of energy production occurred 20 hours later suggestive of metabolic preconditioning. Indeed, proteomic profiling showed that oral ibogaine (100 mg/kg) administration to female mice produced changes in spinal cord protein abundance indicative of increasing metabolic stress from 0.25-24 hours that resolved by 72 hours. This was accompanied by activation, inhibition and reactivation of synaptic plasticity and neural repair signaling pathways at 0.25, 4-24 and 72 hours, respectively. Hence, by inducing transient metabolic stress, ibogaine may re-open a neurodevelopmental window characterized by enhanced synaptic plasticity and neural repair. Ibogaine binds with high affinity to the sigma-2 receptor. Like mitoKv1-3 inhibitors and sigma-2 receptor ligands, ibogaine transiently elevated AMP-activated protein kinase (0.25 hours) and temporally suppressed mechanistic target of rapamycin and lipid synthesis pathways (0.25-24 hours) implicated in enhanced synaptic plasticity and neural repair. These findings suggest ibogaine rewires and repairs neural circuits by a metabolic preconditioning mechanism, perhaps involving mitoKv1.3 and the sigma-2 receptor.

Replication and transcription of Mononegavirales (MNV) occur in cytoplasmic viral factories. In rabies virus (RABV) infected cells, these membrane-less, liquid-liquid phase-separated organelles are called Negri bodies (NBs). Their accumulation of viral mRNAs suggests that, in addition to supporting viral RNA synthesis, they may also serve as sites of viral protein translation. Using immunofluorescence and confocal microscopy, we demonstrate that ribosomal subunit proteins and translation initiation factors are excluded from NBs, including the ribosomal protein RPL40, a component of the large ribosomal subunit involved in the unconventional translation initiation mechanism of RABV. To investigate the spatiotemporal dynamics of protein synthesis during RABV infection, we performed pulse-labeling experiments with the methionine analogue homopropargylglycine (HPG). HPG labeling revealed a rapid accumulation of newly synthesized proteins within NBs, occurring independently of both microtubule and actin cytoskeletons. RABV infection also triggered the formation of stress granules (SGs), in which some ribosomal proteins and translation initiation factors are concentrated. Notably, a marked inhibition of protein synthesis was observed in SG-positive cells. Together, these findings indicate that in RABV infected cells translation occurs outside NBs, followed by the targeting of nascent viral proteins to these compartments.

Understanding how photosynthetic organisms acclimate to excess light is essential for the development of robust chassis for synthetic biology approaches aimed at expanding the photosynthetically active absorption spectrum. High-light (HL) tolerant strains were previously generated by laboratory evolution from a laboratory type (LT) strain of Synechocystis sp. PCC 6803, with tolerance attributed to a small number of specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1F124L) and translation elongation factor G2 (EF-G2R461C). Reintroduction of these mutations into the LT background was sufficient to confer HL tolerance. However, the mechanisms by which a limited set of point mutations mediates HL tolerance have remained unclear. Here, integrated transcriptomic and proteomic analyses of HL-tolerant strains reveal a coordinated network of responses underlying HL tolerance. The NdhF1F124L mutation increased the accumulation of NDH-1 complex subunits, likely accounting for the previously observed enhancement of cyclic electron flow. In contrast, EF-G2R461C increased the abundance of multiple functional classes of proteins associated with HL tolerance, while post-transcriptionally reducing the level of the phycobilisome linker protein CpcC2, resulting in a decreased antenna size. Integrated analyses further demonstrated that HL tolerance involves transcriptional regulation of protein abundance, including the maintenance of phosphate metabolism. Consistently, overexpression of two genes from the Pho regulon increased HL tolerance. Overall, this study demonstrates how a small number of point mutations in genes with central cellular functions can reprogram the cyanobacterial cell to achieve enhanced tolerance to HL.

The maximum growth rate of microbes plays a central role in shaping ecological outcomes and is a major target of bioengineering efforts. Previous data compilations suggest single cell maximum growth rates mostly decrease with cell volume across species, except for an initial increase from the very smallest microbes to an intermediate size. Here we hypothesized that this unimodal relationship is shaped by the volumetric requirements of ribosomes and the surface area-dependence of nutrient supply. We developed a mechanistic model relating ribosomal protein mass fraction to growth rate and cell size. Predictions from this model were empirically supported by proteomic data from 97 prokaryotes ranging over 3 orders of magnitude in cell volume. Our analysis suggests that the smallest cells' growth rates are restricted by the volumetric constraints on ribosomes per cell and rely on compact, heterotrophic metabolisms. In contrast, growth rates of the largest prokaryotes face geometric constraints on specific resource acquisition rates, capping growth rates and investment in ribosomal protein, but allowing investment in protein-expensive metabolisms including phototrophy and sulfur oxidation. Prokaryotic cells of intermediate size face neither constraint and exhibit a higher maximum growth rate potential, which, we hypothesize, is associated with elevated metabolic diversity.

Bacterial cultures entering stationary phase (SP) undergo complex physiological alterations, helping the cells to survive when medium resources are exhausted. The onset of various stress responses underlying SP physiology is generally believed to be due to reactions of individual cells to the environmental cues such as starvation, toxic metabolites, etc. The SP physiological state makes bacteria unsuitable for the replication of most bacteriophages; however, some phages are able to infect and multiply in them. We investigated Escherichia coli phage DH23 growth in SP cultures of E. coli MG1655 infected at different hours post inoculation (ages). Under our conditions the cells enter SP at about 6 h, but the phage replication was possible till 11h and then dropped abruptly by 13h of culture aging, indicating an abrupt physiological change to deeper dormancy during SP. The onset of this phage-inhibiting middle-SP dormancy (MSPD) turned out to be mediated by intercellular communication mediated by the middle-SP signal particles (MSP) which are larger than 100 kDa and contain both RNA and DNA. These MSP are inactivated by RNAse or by DNAse, enabling phage growth in 14h-old cultures and lifts the tolerance of such cultures to kanamycin. Moreover, the RNAse or DNAse treatment makes 24h culture spent media suitable for additional cell growth, and cultures initiated in fresh LB supplemented with RNAse reach an OD600 about 1.5 times higher compared to the untreated control. This indicates that MSP signaling influences cell physiology well before the MSPD onset and helps the population cease growth pre-emptively to save about 1/3 of medium resources to support the viability during SP.

Virophages are small double-stranded DNA viruses that co-infect eukaryotic cells along with giant viruses. They are widespread in aquatic ecosystems and can influence microbial population dynamics by acting as an adaptive antiviral defense system, limiting production of giant virus progeny. Giant viruses extensively reprogram host metabolism to support their replication, yet little is known about how this process is altered by virophages. Characterizing these alterations may reveal the metabolic mechanisms underlying virophage-mediated changes in infection outcomes. Here, we used one of the few established host-giant virus-virophage experimental systems, consisting of the marine heterotrophic flagellate Cafeteria burkhardae, its giant virus CroV, and the virophage mavirus, to investigate lipid remodeling during viral infection and virophage co-infection. We found that infection by the giant virus strongly induced triacylglycerols containing furan fatty acids (FuFAs), a class of potent antioxidants that may protect infected cells and newly formed virions from oxidative damage. These lipids were also detected in purified giant virus particles, linking their accumulation to virus production. Co-infection with the virophage reduced the FuFA response and was instead characterized by pronounced accumulation of the betaine lipid diacylglyceryltrimethylhomoserine (DGTS). Most of the induced DGTS species were also present in the giant virus particles, suggesting that their accumulation during co-infection may be associated with altered giant virus production and lipid utilization. Together, these findings show that virophage co-infection reshapes giant virus-induced remodeling of the host lipidome, revealing a potential metabolic basis for their effects on virus production and infection outcomes.

The 5'-untranslated region (5'-UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly structured and comprises five conserved stem-loops that serve as cis-acting regulatory elements. During infection, host proteins are recruited to these elements and regulate multiple steps during the viral life cycle. Stem-loop 5 (SL5) is highly conserved among different alpha- and betacoronaviruses and includes the start codon of non-structural protein 1 (nsp1). However, the function of SL5 in viral replication and its interacting proteins remain to be determined. Here, we identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a host protein functionally interacting with SL5. HNRNPM translocates into the cytoplasm during SARS-CoV-2 infection and promotes protein synthesis from the viral 5'-UTR. Mutations in the lower stem of SL5 impair HNRNPM binding and reduce protein synthesis. Notably, the interaction between HNRNPM and SL5 is conserved among different alpha- and betacoronaviruses. Our findings indicate that HNRNPM is an important host factor that regulates viral translation to promote SARS-CoV-2 replication.

Unlike most other heterotrophic protist lineages which synthesize glycogen, alveolates accumulate -glucan storage polysaccharides in the form of starch. Within Alveolata, ciliates do so by using a simple pathway analogous to that used for glycogen metabolism in eukaryotes. However, Myzozoa have evolved five additional enzymatic steps which are common to Dinoflagellata, Chromerids and Apicomplexa. Moreover, dinoflagellates have acquired seven additional enzymes that distinguish them from the Chromerids-Apicompexa lineages, while chromerids possess only a single enzyme not found in dinoflagellates. Such modifications are not found in other eukaryotic lineages save for those resulting directly from primary or secondary plastid endosymbiosis. The many differences affecting the starch pathway of Myzozoa can be explained by a single secondary or tertiary plastid endosymbiosis of a starch-containing alga followed by distinct compartmentalisation outcomes of starch metabolism in Dinoflagellata relative to chromerids and Apicomplexa.

Microbial metabolism in extreme environments has long been shrouded in profound mystery. Distinct from the direct assimilation of exogenous organic carbon, microorganisms predominantly exploit inorganic nitrogen substrates as their primary nitrogen sources. As a critical nitrogen assimilation pathway, assimilatory nitrite reduction plays a vital role in microbial nitrogen acquisition, especially in extreme environments with poor nutrient availability. In this study, we identified the coexistence of two isoforms of NADH-dependent nitrite reductase in single genomes. NIR- and NIR-{beta}, named based on their phylogenetic relationships, exhibit significant differences in protein sequence, genomic context and evolutionary relationship, a phenomenon that has rarely been reported before. They also show distinct expression patterns, which may allow bacteria to achieve efficient nitrogen conversion in nutrient-poor environments. This finding calls for a revision of the conventional model of nitrogen cycling gene combinations inferred from common model microorganisms and holds numerous implications for how we understand and study the environmental adaptation mechanisms of extremophiles.

Adeno-associated viral (AAV) vectors possess immense potential for gene therapy applications. However, microglia can mount an immune response against AAV vectors. Yet, microglia are therapeutic targets since they play a driving role in the progression of neurodegenerative diseases. Thus, it is critical to understand the cellular processes driving the microglia-mediated immune response against AAV vectors. To this end, we modelled AAV6 induced inflammation upon retinal gene therapy in minipigs. Upon AAV6 administration, the IBA1+ cells demonstrated a morphological transition to the amoeboid form, indicating microglial reactivity. We also observed the invasion of peripheral immune cells in the retina. To elucidate the underlying molecular mechanisms, we transduced human iPSC derived microglia-like cells (iMGLs) in vitro with AAV6 and AAV8 respectively. Only AAV6 transduced iMGLs exhibited morphological features and a transcriptomic signature of microglial reactivity. The magnitude of intracellular AAV6 viral genomes was significantly higher as compared to the AAV8 condition. The elevated levels of intracellular AAV6 genomes induced a DNA damage response in AAV6-transduced iMGLs, which is a key feature of neuroinflammation-linked microglial reactivity. Thus, our work highlights the impact of intracellular viral genome accumulation on microglial reactivity and its implications for retinal gene therapy associated inflammation.