Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a transcriptional coactivator involved in stress response, autoimmune disease, cancer and HIV replication. A fusion between the nuclear pore protein NUP98 and LEDGF/p75 has been found in human acute and chronic myeloid leukemia and association of LEDGF/p75 with mixed-lineage leukemia (MLL)/menin is critical for leukemic transformation. During lentiviral replication, LEDGF/p75 tethers the pre-integration complex to the host chromatin resulting in a bias of integration into active transcription units (TUs). The consensus function of LEDGF/p75 is tethering of cargos to chromatin. In this regard, we determined the LEDGF/p75 chromatin binding profile. To this purpose, we used DamID technology and focused on the highly annotated ENCODE (Encyclopedia of DNA Elements) regions. LEDGF/p75 primarily binds downstream of the transcription start site of active TUs in agreement with the enrichment of HIV-1 integration sites at these locations. We show that LEDGF/p75 binding is not restricted to stress response elements in the genome, and correlation analysis with more than 200 genomic features revealed an association with active chromatin markers, such as H3 and H4 acetylation, H3K4 monomethylation and RNA polymerase II binding. Interestingly, some associations did not correlate with HIV-1 integration indicating that not all LEDGF/p75 complexes on the chromosome are amenable to HIV-1 integration.

Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown.

Loss-of-function mutations in the PINK1 gene lead to recessive forms of Parkinson's disease. Animal models with depleted PINK1 expression have failed to reproduce significant nigral dopaminergic neurodegeneration and clear alpha-synuclein pathology, main characteristics of the disease. In this study, we investigated whether alpha-synuclein pathology is altered in the absence of PINK1 in cell culture and in vivo. We observed that downregulation of PINK1 enhanced alpha-synuclein aggregation and apoptosis in a neuronal cell culture model for synucleinopathy. Silencing of PINK1 expression in mouse substantia nigra using recombinant adeno-associated viral vectors did not induce dopaminergic neurodegeneration in a long-term study up to 10 months, nor did it enhance or accelerate dopaminergic neurodegeneration after alpha-synuclein overexpression. However, in PINK1 knockout mice, overexpression of alpha-synuclein in the substantia nigra resulted in enhanced dopaminergic neurodegeneration as well as significantly higher levels of alpha-synuclein phosphorylation at serine 129 at 4 weeks postinjection. In conclusion, our results demonstrate that total loss of PINK1 leads to an increased sensitivity to alpha-synuclein-induced neuropathology and cell death in vivo.

The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.

The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.

LEDGINs are novel allosteric HIV integrase (IN) inhibitors that target the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. They block HIV-1 integration by abrogating the interaction between LEDGF/p75 and IN as well as by allosterically inhibiting the catalytic activity of IN.

Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra.

Our understanding on how selective mitochondrial autophagy, or mitophagy, can sustain the archetypal properties of stem cells is incomplete. PTEN-induced putative kinase 1 (PINK1) plays a key role in the maintenance of mitochondrial morphology and function and in the selective degradation of damaged mitochondria by mitophagy. Here, using embryonic fibroblasts fromPINK1 gene-knockout (KO) mice, we evaluated whether mitophagy is a causal mechanism for the control of cell-fate plasticity and maintenance of pluripotency. Loss of PINK1-dependent mitophagy was sufficient to dramatically decrease the speed and efficiency of induced pluripotent stem cell (iPSC) reprogramming. Mitophagy-deficient iPSC colonies, which were characterized by a mixture of mature and immature mitochondria, seemed unstable, with a strong tendency to spontaneously differentiate and form heterogeneous populations of cells. Although mitophagy-deficient iPSC colonies normally expressed pluripotent markers, functional monitoring of cellular bioenergetics revealed an attenuated glycolysis in mitophagy-deficient iPSC cells. Targeted metabolomics showed a notable alteration in numerous glycolysis- and TCA-related metabolites in mitophagy-deficient iPSC cells, including a significant decrease in the intracellular levels of α-ketoglutarate -a key suppressor of the differentiation path in stem cells. Mitophagy-deficient iPSC colonies exhibited a notably reduced teratoma-initiating capacity, but fully retained their pluripotency and multi-germ layer differentiation capacity in vivo. PINK1-dependent mitophagy pathway is an important mitochondrial switch that determines the efficiency and quality of somatic reprogramming. Mitophagy-driven mitochondrial rejuvenation might contribute to the ability of iPSCs to suppress differentiation by directing bioenergetic transition and metabolome remodeling traits. These findings provide new insights into how mitophagy might influence the stem cell decisions to retain pluripotency or differentiate in tissue regeneration and aging, tumor growth, and regenerative medicine.

Vaccination against measles, mumps, and rubella (MMR) and yellow fever (YF) with live attenuated viruses can rarely cause life-threatening disease. Severe illness by MMR vaccines can be caused by inborn errors of type I and/or III interferon (IFN) immunity (mutations in IFNAR2, STAT1, or STAT2). Adverse reactions to the YF vaccine have remained unexplained. We report two otherwise healthy patients, a 9-yr-old boy in Iran with severe measles vaccine disease at 1 yr and a 14-yr-old girl in Brazil with viscerotropic disease caused by the YF vaccine at 12 yr. The Iranian patient is homozygous and the Brazilian patient compound heterozygous for loss-of-function IFNAR1 variations. Patient-derived fibroblasts are susceptible to viruses, including the YF and measles virus vaccine strains, in the absence or presence of exogenous type I IFN. The patients' fibroblast phenotypes are rescued with WT IFNAR1 Autosomal recessive, complete IFNAR1 deficiency can result in life-threatening complications of vaccination with live attenuated measles and YF viruses in previously healthy individuals.

IKAROS (encoded by IKZF1) is an important hematopoietic transcription factor critical for early B cell differentiation, with major defects known to lead to low B cell numbers and hypogammaglobulinemia. More perplexing is the link between IKZF1 variants and autoimmunity, including polymorphisms associated with susceptibility to SLE, and recently, rare variants driving monogenic autoimmunity. We identified a novel p.L188V mutation in IKZF1 in the index patient and her father and found this mutation to lead to loss of DNA binding. Peripheral B cells lacking a full complement of IKAROS function show upregulation of molecules accentuating B cell activation, while CD22, a key negative feedback circuit, is suppressed. The resulting hyperresponsiveness of peripheral B cells, in combination with elevated follicular helper T cell (Tfh) numbers, provides a putative mechanistic explanation for the association of IKZF1 variants with the emergence of autoimmune manifestations in this kindred.

The bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension and mutations in the BMPR2 gene have been observed in both heritable and idiopathic pulmonary arterial hypertension. However, all BMPR2 mutation carriers do not develop pulmonary arterial hypertension, and inflammation could trigger the development of the disease in BMPR2 mutation carriers. Circulating levels and/or lung tissue expression of cytokines such as tumor necrosis factor-α or interleukin-18 are elevated in patients with pulmonary arterial hypertension and could be involved in the pathogenesis of pulmonary arterial hypertension. We consequently hypothesized that cytokines could trigger endothelial dysfunction in addition to impaired BMPRII signaling. Our aim was to determine whether impairment of BMPRII signaling might affect endothelium barrier function and adhesiveness to monocytes, in response to cytokines. BMPR2 was silenced in human lung microvascular endothelial cells (HLMVECs) using lentiviral vectors encoding microRNA-based hairpins. Effects of tumor necrosis factor-α and interleukin-18 on HLMVEC adhesiveness to the human monocyte cell line THP-1, adhesion molecule expression, endothelial barrier function and activation of P38MAPK were investigated in vitro. Stable BMPR2 silencing in HLMVECs resulted in impaired endothelial barrier function and constitutive activation of P38MAPK. Adhesiveness of BMPR2-silenced HLMVECs to THP-1 cells was enhanced by tumor necrosis factor-α and interleukin-18 through ICAM-1 adhesion molecule. Interestingly, tumor necrosis factor-α induced activation of P38MAPK and disrupted endothelial barrier function in BMPR2-silenced HLMVECs. Altogether, our findings showed that stable BMPR2 silencing resulted in impaired endothelial barrier function and activation of P38MAPK in HLMVECs. In BMPR2-silenced HLMVECs, cytokines enhanced adhesiveness capacities, activation of P38MAPK and impaired endothelial barrier function suggesting that cytokines could trigger the development of pulmonary arterial hypertension in a context of impaired BMPRII signaling pathway.

Synucleinopathies, such as Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are defined by the presence of α-synuclein (αSYN) aggregates throughout the nervous system but diverge from one another with regard to their clinical and pathological phenotype. The recent generation of pure fibrillar αSYN polymorphs with noticeable differences in structural and phenotypic traits has led to the hypothesis that different αSYN strains may be in part responsible for the heterogeneous nature of synucleinopathies. To further characterize distinct αSYN strains in the human brain, and establish a structure-pathology relationship, we pursued a detailed comparison of αSYN assemblies derived from well-stratified patients with distinct synucleinopathies. We exploited the capacity of αSYN aggregates found in the brain of patients suffering from PD, MSA or DLB to seed and template monomeric human αSYN in vitro via a protein misfolding cyclic amplification assay. A careful comparison of the properties of total brain homogenates and pure in vitro amplified αSYN fibrillar assemblies upon inoculation in cells and in the rat brain demonstrates that the intrinsic structure of αSYN fibrils dictates synucleinopathies characteristics. We report that MSA strains show several similarities with PD strains, but are significantly more potent in inducing motor deficits, nigrostriatal neurodegeneration, αSYN pathology, spreading, and inflammation, reflecting the aggressive nature of this disease. In contrast, DLB strains display no or only very modest neuropathological features under our experimental conditions. Collectively, our data demonstrate a specific signature for PD, MSA, and DLB-derived strains that differs from previously described recombinant strains, with MSA strains provoking the most aggressive phenotype and more similarities with PD compared to DLB strains.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections, and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical IFNγ activation site (GAS) sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion, our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism toward transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.

To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLVW390A-CBX was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLVW390A-CBX vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLVW390A-CBX-EFS produced like MLVWT and efficiently transduced laboratory cells and primary human CD34+ hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLVWT transduction, while this remained unaffected when applying MLVW390A-CBX. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLVW390A-CBX-EFS. Taken together, our results underscore the potential of MLVW390A-CBX-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.

The link between the gut and the brain in Parkinson's disease (PD) pathogenesis is currently a subject of intense research. Indeed, gastrointestinal dysfunction is known as an early symptom in PD and inflammatory bowel disease (IBD) has recently been recognised as a risk factor for PD. The leucine-rich repeat kinase 2 (LRRK2) is a PD- and IBD-related protein with highest expression in immune cells. In this study, we provide evidence for a central role of LRRK2 in gut inflammation and PD. The presence of the gain-of-function G2019S mutation significantly increases the disease phenotype and inflammatory response in a mouse model of experimental colitis based on chronic dextran sulphate sodium (DSS) administration. Bone marrow transplantation of wild-type cells into G2019S knock-in mice fully rescued this exacerbated response, proving the key role of mutant LRRK2 in immune cells in this experimental colitis model. Furthermore, partial pharmacological inhibition of LRRK2 kinase activity also reduced the colitis phenotype and inflammation. Moreover, chronic experimental colitis also induced neuroinflammation and infiltration of peripheral immune cells into the brain of G2019S knock-in mice. Finally, combination of experimental colitis with overexpression of α-synuclein in the substantia nigra aggravated motor deficits and dopaminergic neurodegeneration in G2019S knock-in mice. Taken together, our results link LRRK2 with the immune response in colitis and provide evidence that gut inflammation can impact brain homeostasis and contribute to neurodegeneration in PD.

Vaginal birth causes pelvic floor injury which may lead to urinary incontinence. Cell therapy has been proposed to assist in functional recovery. We aim to assess if intra-arterial injection of rat mesoangioblasts (MABs) and stable Vascular Endothelial Growth Factor (VEGF)-expressing MABs, improve recovery of urethral and vaginal function following simulated vaginal delivery (SVD). Female rats (n = 86) were assigned to either injection of saline (control), allogeneic-MABs (MABsallo), autologous-MABs (MABsauto) or allogeneic-MABs transduced to stably expressed VEGF (MABsallo-VEGF). One hour after SVD, 0.5 × 106 MABs or saline were injected into the aorta. Primary outcome was urethral (7d and 14d) and vaginal (14d) function; others were bioluminescent imaging for cell tracking (1, 3 and 7d), morphometry (7, 14 and 60d) and mRNAseq (3 and 7d). All MABs injected rats had external urethral sphincter and vaginal function recovery within 14d, as compared to only half of saline controls. Functional recovery was paralleled by improved muscle regeneration and microvascularization. Recovery rate was not different between MABsallo and MABsauto. MABsallo-VEGF accelerated functional recovery and increased GAP-43 expression at 7d. At 3d we detected major transcriptional changes in the urethra of both MABsallo and MABsallo-VEGF-injected animals, with upregulation of Rho/GTPase activity, epigenetic factors and dendrite development. MABSallo also upregulated transcripts that encode proteins involved in myogenesis and downregulated pro-inflammatory processes. MABsallo-VEGF also upregulated transcripts that encode proteins involved in neuron development and downregulated genes involved in hypoxia and oxidative stress. At 7d, urethras of MABsallo-VEGF-injected rats showed downregulation of oxidative and inflammatory response compared to MABSallo. Intra-arterial injection of MABsallo-VEGF enhances neuromuscular regeneration induced by untransduced MABs and accelerates the functional urethral and vaginal recovery after SVD.

This study compared the expression pattern, laminar distribution, and cell specificity of several rAAV serotypes (2/1, 2/5, 2/7, 2/8, and 2/9) injected in the primary visual cortex (V1) of adult C57Bl/6J mice. In order to obtain specific expression in certain neuron subtypes, different promoter sequences were evaluated for excitatory cell specificity: a universal cytomegalovirus (CMV) promoter, and two versions of the excitatory neuron-specific Ca(2+) /calmodulin-dependent kinase subunit α (CaMKIIα) promoter, CaMKIIα 0.4 and CaMKIIα 1.3. The spatial distribution as well as the cell type specificity was immunohistochemically verified. Depending on the rAAV serotype used, the transduced volume expressing reporter protein differed substantially (rAAV2/5 ≫ 2/7 ≈ 2/9 ≈ 2/8 ≫ 2/1). Excitatory neuron-specific targeting was promoter-dependent, with a surprising difference between the 1.3 kb and 0.4 kb CaMKIIα promoters. While CaMKIIα 1.3 and CMV carrying vectors were comparable, with 78% of the transduced neurons being excitatory for CMV and 82% for CaMKIIα 1.3, the shorter CaMKIIα 0.4 version resulted in 95% excitatory specificity. This study therefore puts forward the CaMKIIα 0.4 promoter as the best choice to target excitatory neurons with rAAVs. Together, these results can be used as an aid to select the most optimal vector system to deliver transgenes into specific rodent neocortical circuits, allowing further elucidation of their functions.

Alpha-synuclein is a key protein in the pathogenesis of Parkinson's disease. Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson's disease, probably through a loss-of-function mechanism. However, the molecular mechanism by which loss of parkin function leads to the development of the disease and the role of alpha-synuclein in parkin-associated Parkinson's disease is still not elucidated. Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach. In this study, we investigated the effect of loss of parkin on alpha-synuclein neuropathology and toxicity in adult rodent brain using viral vectors. Therefore, we overexpressed human wild type alpha-synuclein in the substantia nigra of parkin knockout and wild type mice using two different doses of recombinant adeno-associated viral vectors.