DOT1L activity affects neural stem cell division mode and reduces differentiation and ASNS expression.

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

Activation of Src family kinase ameliorates secretory trafficking in mutant prion protein cells.

Fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and Gerstmann-Sträussler-Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.

Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity.

Prion protein (PrP) mutations are linked to genetic prion diseases, a class of phenotypically heterogeneous neurodegenerative disorders with invariably fatal outcome. How mutant PrP triggers neurodegeneration is not known. Synaptic dysfunction precedes neuronal loss but it is not clear whether, and through which mechanisms, disruption of synaptic activity ultimately leads to neuronal death. Here we show that mutant PrP impairs the secretory trafficking of AMPA receptors (AMPARs). Specifically, intracellular retention of the GluA2 subunit results in synaptic exposure of GluA2-lacking, calcium-permeable AMPARs, leading to increased calcium permeability and enhanced sensitivity to excitotoxic cell death. Mutant PrPs linked to different genetic prion diseases affect AMPAR trafficking and function in different ways. Our findings identify AMPARs as pathogenic targets in genetic prion diseases, and support the involvement of excitotoxicity in neurodegeneration. They also suggest a mechanistic explanation for how different mutant PrPs may cause distinct disease phenotypes.

Correction: Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity.

[This corrects the article DOI: 10.1371/journal.ppat.1008654.].

Doxycycline rescues recognition memory and circadian motor rhythmicity but does not prevent terminal disease in fatal familial insomnia mice.

Fatal familial insomnia (FFI) is a dominantly inherited prion disease linked to the D178N mutation in the gene encoding the prion protein (PrP). Symptoms, including insomnia, memory loss and motor abnormalities, appear around 50 years of age, leading to death within two years. No treatment is available. A ten-year clinical trial of doxycycline (doxy) is under way in healthy individuals at risk of FFI to test whether presymptomatic doxy prevents or delays the onset of disease. To assess the drug's effect in a tractable disease model, we used Tg(FFI-26) mice, which accumulate aggregated and protease-resistant PrP in their brains and develop a fatal neurological illness highly reminiscent of FFI. Mice were treated daily with 10 mg/kg doxy starting from a presymptomatic stage for twenty weeks. Doxy rescued memory deficits and restored circadian motor rhythmicity in Tg(FFI-26) mice. However, it did not prevent the onset and progression of motor dysfunction, clinical signs and progression to terminal disease. Doxy did not change the amount of aggregated and protease-resistant PrP, but reduced microglial activation in the hippocampus. Presymptomatic doxy treatment rescues cognitive impairment and the motor correlates of sleep dysfunction in Tg(FFI-26) mice but does not prevent fatal disease.

C. elegans detects toxicity of traumatic brain injury generated tau.

Traumatic brain injury (TBI) is associated with widespread tau pathology in about 30% of patients surviving late after injury. We previously found that TBI in mice induces the formation of an abnormal form of tau (tauTBI) which progressively spreads from the site of injury to remote brain regions. Intracerebral inoculation of TBI brain homogenates into naïve mice induced progressive tau pathology, synaptic loss and late cognitive decline, suggesting a pivotal role of tauTBI in post-TBI neurodegeneration. However, the possibility that tauTBI was a marker of TBI-associated neurodegeneration rather than a toxic driver of functional decline could not be excluded. Here we employed the nematode C. elegans as a biosensor to test the pathogenic role of TBI generated tau. The motility of this nematode depends on efficient neuromuscular transmission and is exceptionally sensitive to the toxicity of amyloidogenic proteins, providing a tractable model for our tests. We found that worms exposed to brain homogenates from chronic but not acute TBI mice, or from mice in which tauTBI had been transmitted by intracerebral inoculation, had impaired motility and neuromuscular synaptic transmission. Results were similar when worms were given brain homogenates from transgenic mice overexpressing tau P301L, a tauopathy mouse model, suggesting that TBI-induced and mutant tau have similar toxic properties. P301L brain homogenate toxicity was similar in wild-type and ptl-1 knock-out worms, indicating that the nematode tau homolog protein PTL-1 was not required to mediate the toxic effect. Harsh protease digestion to eliminate the protein component of the homogenates, pre-incubation with anti-tau antibodies or tau depletion by immunoprecipitation, abolished the toxicity. Homogenates of chronic TBI brains from tau knock-out mice were not toxic to C. elegans, whereas oligomeric recombinant tau was sufficient to impair their motility. This study indicates that tauTBI impairs motor activity and synaptic transmission in C. elegans and supports a pathogenic role of tauTBI in the long-term consequences of TBI. It also sets the groundwork for the development of a C. elegans-based platform for screening anti-tau compounds.

Proteomic Analysis of Marinesco-Sjogren Syndrome Fibroblasts Indicates Pro-Survival Metabolic Adaptation to SIL1 Loss.

Marinesco-Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.

PERK inhibition delays neurodegeneration and improves motor function in a mouse model of Marinesco-Sjögren syndrome.

Marinesco-Sjögren syndrome (MSS) is a rare, early onset, autosomal recessive multisystem disorder characterized by cerebellar ataxia, cataracts and myopathy. Most MSS cases are caused by loss-of-function mutations in the gene encoding SIL1, a nucleotide exchange factor for the molecular chaperone BiP which is essential for correct protein folding in the endoplasmic reticulum. Woozy mice carrying a spontaneous Sil1 mutation recapitulate key pathological features of MSS, including cerebellar atrophy with degeneration of Purkinje cells and progressive myopathy. Because the PERK branch of the unfolded protein response is activated in degenerating neurons of woozy mice, and inhibiting PERK-mediated translational attenuation has shown protective effects in protein-misfolding neurodegenerative disease models, we tested the therapeutic efficacy of GSK2606414, a potent inhibitor of PERK. Mice were chronically treated with GSK2606414 starting from a presymptomatic stage, and the effects were evaluated on biochemical, histopathological and clinical readouts. GSK2606414 delayed Purkinje cell degeneration and the onset of motor deficits, prolonging the asymptomatic phase of the disease; it also reduced the skeletal muscle abnormalities and improved motor performance during the symptomatic phase. The protein but not the mRNA level of ORP150, a nucleotide exchange factor which can substitute for SIL1, was increased in the cerebellum of GSK2606414-treated woozy mice, suggesting that translational recovery promoted the synthesis of this alternative BiP co-factor. Targeting PERK signaling may have beneficial disease-modifying effects in carriers of SIL1 mutations.

Cellular prion protein neither binds to alpha-synuclein oligomers nor mediates their detrimental effects.

α-Synuclein oligomers are crucial players in the pathogenesis of Parkinson's disease. Some mechanisms involved in α-synuclein oligomer detrimental effects include membrane damage, neuroinflammation and protein-protein interactions. Recently, the cellular prion protein (PrPC) emerged as an interactor of α-synuclein oligomers, apparently mediating their detrimental activities. Through direct in vivo and in vitro approaches we herein investigated the existence of a direct cross-talk between α-synuclein oligomers and PrPC. In vitro, we assessed α-synuclein oligomer toxicity by comparing the effect in Prnp+/+ versus PrPC knockout (Prnp0/0) hippocampal neurons. Through an in vivo acute mouse model, where α-synuclein oligomers injected intracerebroventricularly induce memory impairment and neuroinflammation, we verified whether these detrimental effects were preserved in Prnp0/0 mice. In addition, PrPC-α-synuclein oligomer direct binding was investigated through surface plasmon resonance. We found that PrPC was not mandatory to mediate α-synuclein oligomer detrimental effects in vitro or in vivo. Indeed, α-synuclein oligomer toxicity was comparable in Prnp+/+ and Prnp0/0 neurons and both Prnp+/+ and Prnp0/0 mice injected with α-synuclein oligomers displayed memory deficit and hippocampal gliosis. Moreover, surface plasmon resonance analyses ruled out PrPC-α-synuclein oligomer binding. Our findings indicate that PrPC neither binds α-synuclein oligomers nor mediates their detrimental actions. Therefore, it is likely that PrPC-dependent and PrPC-independent pathways co-exist in Parkinson's disease.

Calcineurin Controls Expression of EAAT1/GLAST in Mouse and Human Cultured Astrocytes through Dynamic Regulation of Protein Synthesis and Degradation.

Alterations in the expression of glutamate/aspartate transporter (GLAST) have been associated with several neuropathological conditions including Alzheimer's disease and epilepsy. However, the mechanisms by which GLAST expression is altered are poorly understood. Here we used a combination of pharmacological and genetic approaches coupled with quantitative PCR and Western blot to investigate the mechanism of the regulation of GLAST expression by a Ca2+/calmodulin-activated phosphatase calcineurin (CaN). We show that treatment of cultured hippocampal mouse and fetal human astrocytes with a CaN inhibitor FK506 resulted in a dynamic modulation of GLAST protein expression, being downregulated after 24-48 h, but upregulated after 7 days of continuous FK506 (200 nM) treatment. Protein synthesis, as assessed by puromycin incorporation in neo-synthesized polypeptides, was inhibited already after 1 h of FK506 treatment, while the use of a proteasome inhibitor MG132 (1 µM) shows that GLAST protein degradation was only suppressed after 7 days of FK506 treatment. In astrocytes with constitutive genetic ablation of CaN both protein synthesis and degradation were significantly inhibited. Taken together, our data suggest that, in cultured astrocytes, CaN controls GLAST expression at a posttranscriptional level through regulation of GLAST protein synthesis and degradation.

Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival in a Mouse Model of Amyotrophic Lateral Sclerosis.

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1G93A mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1G93A mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENT We provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1G93A mouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.

Transgenic fatal familial insomnia mice indicate prion infectivity-independent mechanisms of pathogenesis and phenotypic expression of disease.

Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD178) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD178. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.

A tetracationic porphyrin with dual anti-prion activity.

Prions are deadly infectious agents made of PrPSc, a misfolded variant of the cellular prion protein (PrPC) which self-propagates by inducing misfolding of native PrPC. PrPSc can adopt different pathogenic conformations (prion strains), which can be resistant to potential drugs, or acquire drug resistance, hampering the development of effective therapies. We identified Zn(II)-BnPyP, a tetracationic porphyrin that binds to distinct domains of native PrPC, eliciting a dual anti-prion effect. Zn(II)-BnPyP binding to a C-terminal pocket destabilizes the native PrPC fold, hindering conversion to PrPSc; Zn(II)-BnPyP binding to the flexible N-terminal tail disrupts N- to C-terminal interactions, triggering PrPC endocytosis and lysosomal degradation, thus reducing the substrate for PrPSc generation. Zn(II)-BnPyP inhibits propagation of different prion strains in vitro, in neuronal cells and organotypic brain cultures. These results identify a PrPC-targeting compound with an unprecedented dual mechanism of action which might be exploited to achieve anti-prion effects without engendering drug resistance.

Erratum: A tetracationic porphyrin with dual anti-prion activity.

[This corrects the article DOI: 10.1016/j.isci.2023.107480.].

Calcineurin Controls Cellular Prion Protein Expression in Mouse Astrocytes.

Prion diseases arise from the conformational conversion of the cellular prion protein (PrPC) into a self-replicating prion isoform (PrPSc). Although this process has been studied mostly in neurons, a growing body of evidence suggests that astrocytes express PrPC and are able to replicate and accumulate PrPSc. Currently, prion diseases remain incurable, while downregulation of PrPC represents the most promising therapy due to the reduction of the substrate for prion conversion. Here we show that the astrocyte-specific genetic ablation or pharmacological inhibition of the calcium-activated phosphatase calcineurin (CaN) reduces PrPC expression in astrocytes. Immunocytochemical analysis of cultured CaN-KO astrocytes and isolation of synaptosomal compartments from the hippocampi of astrocyte-specific CaN-KO (ACN-KO) mice suggest that PrPC is downregulated both in vitro and in vivo. The downregulation occurs without affecting the glycosylation of PrPC and without alteration of its proteasomal or lysosomal degradation. Direct assessment of the protein synthesis rate and shotgun mass spectrometry proteomics analysis suggest that the reduction of PrPC is related to the impairment of global protein synthesis in CaN-KO astrocytes. When WT-PrP and PrP-D177N, a mouse homologue of a human mutation associated with the inherited prion disease fatal familial insomnia, were expressed in astrocytes, CaN-KO astrocytes showed an aberrant localization of both WT-PrP and PrP-D177N variants with predominant localization to the Golgi apparatus, suggesting that ablation of CaN affects both WT and mutant PrP proteins. These results provide new mechanistic details in relation to the regulation of PrP expression in astrocytes, suggesting the therapeutic potential of astroglial cells.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

The hydrophobic core region governs mutant prion protein aggregation and intracellular retention.

Approx. 15% of human prion diseases have a pattern of autosomal dominant inheritance, and are linked to mutations in the gene encoding PrP (prion protein), a GPI (glycosylphosphatidylinositol)-anchored protein whose function is not clear. The cellular mechanisms by which PrP mutations cause disease are also not known. Soon after synthesis in the ER (endoplasmic reticulum), several mutant PrPs misfold and become resistant to phospholipase cleavage of their GPI anchor. The biosynthetic maturation of the misfolded molecules in the ER is delayed and, during transit in the secretory pathway, they form detergent-insoluble and protease-resistant aggregates, suggesting that intracellular PrP aggregation may play a pathogenic role. We have investigated the consequence of deleting residues 114-121 within the hydrophobic core of PrP on the aggregation and cellular localization of two pathogenic mutants that accumulate in the ER and Golgi apparatus. Compared with their full-length counterparts, the deleted molecules formed smaller protease-sensitive aggregates and were more efficiently transported to the cell surface and released by phospholipase cleavage. These results indicate that mutant PrP aggregation and intracellular retention are closely related and depend critically on the integrity of the hydrophobic core. The discovery that Delta114-121 counteracts misfolding and improves the cellular trafficking of mutant PrP provides an unprecedented model for assessing the role of intracellular aggregation in the pathogenesis of prion diseases.

Forced diuresis oriented by point-of-care ultrasound in cardiorenal syndrome type 5 due to light chain myeloma-The role of hepatic venogram: A case report.

Monitoring venous congestion by ultrasound assessment of hepatic venogram allowed individualized fluid management in severe cardiorenal syndrome type 5 due to light chain myeloma, preserving residual renal function and avoiding heart failure.

SARS-CoV-2-related ARDS in a maintenance hemodialysis patient: case report on tailored approach by daily hemodialysis, noninvasive ventilation, tocilizumab, anxiolytics, and point-of-care ultrasound.

Without rescue drugs approved, holistic approach by daily hemodialysis, noninvasive ventilation, anti-inflammatory medications, fluid assessment by bedside ultrasound, and anxiolytics improved outcomes of a maintenance hemodialysis patient affected by severe COVID-19.

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons.

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.