Inhibition of microglia overactivation restores neuronal survival in a mouse model of CDKL5 deficiency disorder

open9noFunding: This work was supported by the Telethon foundation (grant number GGP19045 to EC), by the Italian parent associations “CDKL5 insieme verso la cura” to EC and by the CARISBO foundation (grant number 2020.0400 to ST).CDKL5 deficiency disorder (CDD), a severe neurodevelopmental disorder characterized by early onset epilepsy, intellectual disability, and autistic features, is caused by mutations in the CDKL5 gene. Evidence in animal models of CDD showed that absence of CDKL5 negatively affects neuronal survival, as well as neuronal maturation and dendritic outgrowth; however, knowledge of the substrates underlying these alterations is still limited. Neuroinflammatory processes are known to contribute to neuronal dysfunction and death. Recent evidence shows a subclinical chronic inflammatory status in plasma from CDD patients. However, to date, it is unknown whether a similar inflammatory status is present in the brain of CDD patients and, if so, whether this plays a causative or exacerbating role in the pathophysiology of CDD.openGalvani, Giuseppe; Mottolese, Nicola; Gennaccaro, Laura; Loi, Manuela; Medici, Giorgio; Tassinari, Marianna; Fuchs, Claudia; Ciani, Elisabetta; Trazzi, StefaniaGalvani, Giuseppe; Mottolese, Nicola; Gennaccaro, Laura; Loi, Manuela; Medici, Giorgio; Tassinari, Marianna; Fuchs, Claudia; Ciani, Elisabetta; Trazzi, Stefani

Early-onset brain alterations during postnatal development in a mouse model of CDKL5 deficiency disorder

Mutations in the CDKL5 gene are the cause of CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental condition characterized by early-onset epilepsy, motor impairment, intellectual disability, and autistic features. A mouse model of CDD, the Cdkl5 KO mouse, that recapitulates several aspects of CDD symptomology, has helped to highlight brain alterations leading to CDD neurological defects. Studies of brain morphogenesis in adult Cdkl5 KO mice showed defects in dendritic arborization of pyramidal neurons and in synaptic connectivity, a hypocellularity of the hippocampal dentate gyrus, and a generalized microglia over-activation. Nevertheless, no studies are available regarding the presence of these brain alterations in Cdkl5 KO pups, and their severity in early stages of life compared to adulthood. A deeper understanding of the CDKL5 deficient brain during an early phase of postnatal development would represent an important milestone for further validation of the CDD mouse model, and for the identification of the optimum time window for treatments that target defects in brain development. In sight of this, we comparatively evaluated the dendritic arborization and spines of cortical pyramidal neurons, cortical excitatory and inhibitory connectivity, microglia activation, and proliferation and survival of granule cells of the hippocampal dentate gyrus in hemizygous Cdkl5 KO male (-/Y) mice aged 7, 14, 21, and 60 days. We found that most of the structural alterations in Cdkl5 -/Y brains are already present in pups aged 7 days and do not worsen with age. In contrast, the difference in the density of excitatory and inhibitory terminals between Cdkl5 -/Y and wild-type mice changes with age, suggesting an age-dependent cortical excitatory/inhibitory synaptic imbalance. Confirming the precocious presence of brain defects, Cdkl5 -/Y pups are characterized by an impairment in neonatal sensory-motor reflexes

Luteolin Treatment Ameliorates Brain Development and Behavioral Performance in a Mouse Model of CDKL5 Deficiency Disorder

CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene, is characterized by early-onset epilepsy, intellectual disability, and autistic features. Although pharmacotherapy has shown promise in the CDD mouse model, safe and effective clinical treatments are still far off. Recently, we found increased microglial activation in the brain of a mouse model of CDD, the Cdkl5 KO mouse, suggesting that a neuroinflammatory state, known to be involved in brain maturation and neuronal dysfunctions, may contribute to the pathophysiology of CDD. The present study aims to evaluate the possible beneficial effect of treatment with luteolin, a natural flavonoid known to have anti-inflammatory and neuroprotective activities, on brain development and behavior in a heterozygous Cdkl5 (+/-) female mouse, the mouse model of CDD that best resembles the genetic clinical condition. We found that inhibition of neuroinflammation by chronic luteolin treatment ameliorates motor stereotypies, hyperactive profile and memory ability in Cdkl5 +/- mice. Luteolin treatment also increases hippocampal neurogenesis and improves dendritic spine maturation and dendritic arborization of hippocampal and cortical neurons. These findings show that microglia overactivation exerts a harmful action in the Cdkl5 +/- brain, suggesting that treatments aimed at counteracting the neuroinflammatory process should be considered as a promising adjuvant therapy for CDD

Treatment with a GSK-3β/HDAC Dual Inhibitor Restores Neuronal Survival and Maturation in an In Vitro and In Vivo Model of CDKL5 Deficiency Disorder

open11noFunding: This research was funded by the Telethon foundation (grant number GGP19045, awarded to E.C.), and by the Italian parent association “CDKL5 insieme verso la cura” (to E.C.).Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a rare neurodevelopmental disorder characterized by early-onset seizures and severe cognitive, motor, and visual impairments. To date there are no therapies for CDKL5 deficiency disorder (CDD). In view of the severity of the neurological phenotype of CDD patients it is widely assumed that CDKL5 may influence the activity of a variety of cellular pathways, suggesting that an approach aimed at targeting multiple cellular pathways simultaneously might be more effective for CDD. Previous findings showed that a single-target therapy aimed at normalizing impaired GSK-3β or histone deacetylase (HDAC) activity improved neurodevelopmental and cognitive alterations in a mouse model of CDD. Here we tested the ability of a first-in-class GSK-3β/HDAC dual inhibitor, Compound 11 (C11), to rescue CDD-related phenotypes. We found that C11, through inhibition of GSK-3β and HDAC6 activity, not only restored maturation, but also significantly improved survival of both human CDKL5-deficient cells and hippocampal neurons from Cdkl5 KO mice. Importantly, in vivo treatment with C11 restored synapse development, neuronal survival, and microglia over-activation, and improved motor and cognitive abilities of Cdkl5 KO mice, suggesting that dual GSK-3β/HDAC6 inhibitor therapy may have a wider therapeutic benefit in CDD patients.openLoi, Manuela; Gennaccaro, Laura; Fuchs, Claudia; Trazzi, Stefania; Medici, Giorgio; Galvani, Giuseppe; Mottolese, Nicola; Tassinari, Marianna; Giorgini, Roberto Rimondini; Milelli, Andrea; Ciani, ElisabettaLoi, Manuela; Gennaccaro, Laura; Fuchs, Claudia; Trazzi, Stefania; Medici, Giorgio; Galvani, Giuseppe; Mottolese, Nicola; Tassinari, Marianna; Giorgini, Roberto Rimondini; Milelli, Andrea; Ciani, Elisabett

Treatment with FRAX486 rescues neurobehavioral and metabolic alterations in a female mouse model of CDKL5 deficiency disorder

Introduction: CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental condition, primarily affecting girls for which no cure currently exists. Neuronal morphogenesis and plasticity impairments as well as metabolic dysfunctions occur in CDD patients. The present study explored the potential therapeutic value for CDD of FRAX486, a brain-penetrant molecule that was reported to selectively inhibit group I p21-activated kinases (PAKs), serine/threonine kinases critically involved in the regulation of neuronal morphology and glucose homeostasis.Methods: The effects of treatment with FRAX486 on CDD-related alterations were assessed in vitro (100 nM for 48h) on primary hippocampal cultures from Cdkl5-knockout male mice (Cdkl5-KO) and in vivo (20 mg/Kg, s.c. for 5 days) on Cdkl5-KO heterozygous females (Cdkl5-Het).Results: The in vitro treatment with FRAX486 completely rescued the abnormal neuronal maturation and the number of PSD95-positive puncta in Cdkl5-KO mouse neurons. In vivo, FRAX486 normalized the general health status, the hyperactive profile and the fear learning defects of fully symptomatic Cdkl5-Het mice. Systemically, FRAX486 treatment normalized the levels of reactive oxidizing species in the whole blood and the fasting-induced hypoglycemia displayed by CdklS-Het mice. In the hippocampus of Cdkl5-Het mice, treatment with FRAX486 rescued spine maturation and PSD95 expression and restored the abnormal PAKs phosphorylation at sites which are critical for their activation (P-PAK-Ser144/141/139) or for the control cytoskeleton remodeling (P-PAK1-Thr212).Conclusions: Present results provide evidence that PAKs may represent innovative therapeutic targets for CDD

Cardiac Functional and Structural Abnormalities in a Mouse Model of CDKL5 Deficiency Disorder

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental disease that mostly affects girls, who are heterozygous for mutations in the X-linked CDKL5 gene. Mutations in the CDKL5 gene lead to a lack of CDKL5 protein expression or function and cause numerous clinical features, including early-onset seizures, marked hypotonia, autistic features, gastrointestinal problems, and severe neurodevelopmental impairment. Mouse models of CDD recapitulate several aspects of CDD symptomology, including cognitive impairments, motor deficits, and autistic-like features, and have been useful to dissect the role of CDKL5 in brain development and function. However, our current knowledge of the function of CDKL5 in other organs/tissues besides the brain is still quite limited, reducing the possibility of broad-spectrum interventions. Here, for the first time, we report the presence of cardiac function/structure alterations in heterozygous Cdkl5 +/- female mice. We found a prolonged QT interval (corrected for the heart rate, QTc) and increased heart rate in Cdkl5 +/- mice. These changes correlate with a marked decrease in parasympathetic activity to the heart and in the expression of the Scn5a and Hcn4 voltage-gated channels. Interestingly, Cdkl5 +/- hearts showed increased fibrosis, altered gap junction organization and connexin-43 expression, mitochondrial dysfunction, and increased ROS production. Together, these findings not only contribute to our understanding of the role of CDKL5 in heart structure/function but also document a novel preclinical phenotype for future therapeutic investigation

Expression of a Secretable, Cell-Penetrating CDKL5 Protein Enhances the Efficacy of Gene Therapy for CDKL5 Deficiency Disorder

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1-S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (\uce\ub3-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (\uce\ub3-H2AXhigh/pATMhigh) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47\ue2\u80\u933.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20\ue2\u80\u933.58). The relationship between the \uce\ub3-H2AXhigh/pATMhigh model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The \uce\ub3-H2AXhigh/pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1 -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (\u3b3-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (\u3b3-H2AXhigh /pATMhigh ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the \u3b3-H2AXhigh /pATMhigh model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The \u3b3-H2AXhigh /pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significanc

Expression of the Hippo transducer TAZ in association with WNT pathway mutations impacts survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy

Background: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Methods: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS). Results: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZpos/WNTmut) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZpos/WNTmutsignature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZpos/WNTmutsignature negatively impacted overall survival. Conclusions: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes