Role of neuroinflammation in the pathophysiology of CDKL5 deficiency disorder

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD), a rare neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene, is characterized by early-onset epilepsy, intellectual disability, and autistic features. To date, little is known about the etiology of CDD and no therapies are available. When overactivated in response to neuronal damage and genetic or environmental factors, microglia – the brain macrophages – cause damage to neighboring neurons by producing neurotoxic factors and pro-inflammatory molecules. Importantly, overactivated microglia have been described in several neurodegenerative and neurodevelopmental disorders, suggesting that active neuroinflammation may account for the compromised neuronal survival and/or brain development observed in these pathologies. Recent evidence shows a subclinical chronic inflammatory status in plasma from CDD patients. However, it is unknown whether a similar inflammatory status is present in the brain of CDD patients and, if so, whether it plays a causative or exacerbating role in the pathophysiology of CDD. Here, we show evidence of a chronic microglia overactivation status in the brain of Cdkl5 KO mice, characterized by alterations in microglial cell number/morphology and increased pro-inflammatory gene expression. We found that the neuroinflammatory process is already present in the postnatal period in Cdkl5 KO mice and worsens during aging. Remarkably, by restoring microglia alterations, treatment with luteolin, a natural anti-inflammatory flavonoid, promotes neuronal survival in the brain of Cdkl5 KO mice since it counteracts hippocampal neuron cell death and protects neurons from NMDA-induced excitotoxic damage. In addition, through the restoration of microglia alterations, luteolin treatment also increases hippocampal neurogenesis and restores dendritic spine maturation and dendritic arborization of hippocampal and cortical pyramidal neurons in Cdkl5 KO mice, leading to improved behavioral performance. These findings highlight new insights into the CDD pathophysiology and provide the first evidence that therapeutic approaches aimed at counteracting neuroinflammation could be beneficial in CDD.

Role of BDNF secretion and signaling in the activity-dependent refinement of synaptic connections

Neuronal networks exhibit diverse types of plasticity, including the activity-dependent regulation of synaptic functions and refinement of synaptic connections. In addition, continuous generation of new neurons in the “adult” brain (adult neurogenesis) represents a powerful form of structural plasticity establishing new connections and possibly implementing pre-existing neuronal circuits (Kempermann et al, 2000; Ming and Song, 2005). Neurotrophins, a family of neuronal growth factors, are crucially involved in the modulation of activity-dependent neuronal plasticity. The first evidence for the physiological importance of this role evolved from the observations that the local administration of neurotrophins has dramatic effects on the activity-dependent refinement of synaptic connections in the visual cortex (McAllister et al, 1999; Berardi et al, 2000; Thoenen, 1995). Moreover, the local availability of critical amounts of neurotrophins appears to be relevant for the ability of hippocampal neurons to undergo long-term potentiation (LTP) of the synaptic transmission (Lu, 2004; Aicardi et al, 2004). To achieve a comprehensive understanding of the modulatory role of neurotrophins in integrated neuronal systems, informations on the mechanisms about local neurotrophins synthesis and secretion as well as ditribution of their cognate receptors are of crucial importance. In the first part of this doctoral thesis I have used electrophysiological approaches and real-time imaging tecniques to investigate additional features about the regulation of neurotrophins secretion, namely the capability of the neurotrophin brain-derived neurotrophic factor (BDNF) to undergo synaptic recycling. In cortical and hippocampal slices as well as in dissociated cell cultures, neuronal activity rapidly enhances the neuronal expression and secretion of BDNF which is subsequently taken up by neurons themselves but also by perineuronal astrocytes, through the selective activation of BDNF receptors. Moreover, internalized BDNF becomes part of the releasable source of the neurotrophin, which is promptly recruited for activity-dependent recycling. Thus, we described for the first time that neurons and astrocytes contain an endocytic compartment competent for BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia. The mechanism of BDNF recycling is reminiscent of that for neurotransmitters and identifies BDNF as a new modulator implicated in neuro- and glio-transmission. In the second part of this doctoral thesis I addressed the role of BDNF signaling in adult hippocampal neurogenesis. I have generated a transgenic mouse model to specifically investigate the influence of BDNF signaling on the generation, differentiation, survival and connectivity of newborn neurons into the adult hippocampal network. I demonstrated that the survival of newborn neurons critically depends on the activation of the BDNF receptor TrkB. The TrkB-dependent decision regarding life or death in these newborn neurons takes place right at the transition point of their morphological and functional maturation Before newborn neurons start to die, they exhibit a drastic reduction in dendritic complexity and spine density compared to wild-type newborn neurons, indicating that this receptor is required for the connectivity of newborn neurons. Both the failure to become integrated and subsequent dying lead to impaired LTP. Finally, mice lacking a functional TrkB in the restricted population of newborn neurons show behavioral deficits, namely increased anxiety-like behavior. These data suggest that the integration and establishment of proper connections by newly generated neurons into the pre-existing network are relevant features for regulating the emotional state of the animal.

Pharmacological Rescue of Dendritic Pathology in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is a genetic pathology characterized by brain hypotrophy and severe cognitive disability. Although defective neurogenesis is an important determinant of cognitive impairment, a severe dendritic pathology appears to be an equally important factor. It is well established that serotonin plays a pivotal role both on neurogenesis and dendritic maturation. Since the serotonergic system is profoundly altered in the DS brain, we wondered whether defects in the hippocampal development can be rescued by treatment with fluoxetine, a selective serotonin reuptake inhibitor and a widely used antidepressant drug. A previous study of our group showed that fluoxetine fully restores neurogenesis in the Ts65Dn mouse model of DS and that this effect is accompanied by a recovery of memory functions. The goal of the current study was to establish whether fluoxetine also restores dendritic development and maturation. In mice aged 45 days, treated with fluoxetine in the postnatal period P3-P15, we examined the dendritic arbor of newborn and mature granule cells of the dentate gyrus (DG). The granule cells of trisomic mice had a severely hypotrophic dendritic arbor, fewer spines and a reduced innervation than euploid mice. Treatment with fluoxetine fully restored all these defects. Moreover the impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons was fully normalized in treated trisomic mice, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The widespread beneficial effects of fluoxetine on the hippocampal formation suggest that early treatment with fluoxetine can be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions. These findings may open the way for future clinical trials in children and adolescents with DS.

Planning and assessment techniques for spine surgeries

The rate of diagnosis and treatment of degenerative spine disorders is increasing, increasing the need for surgical intervention. Posterior spine fusion is one surgical intervention used to treat various spine degeneration pathologies To minimize the risk of complications and provide patients with positive outcomes, preoperative planning and postsurgical assessment are necessary. This PhD aimed to investigate techniques for the surgical planning and assessment of spine surgeries. Three main techniques were assessed: stereophotogrammetric motion analysis, 3D printing of complex spine deformities and finite element analysis of the thoracolumbar spine. Upon reviewing the literature on currently available spine kinematics protocol, a comprehensive motion analysis protocol to measure the multi-segmental spine motion was developed. Using this protocol, the patterns of spine motion in patients before and after posterior spine fixation was mapped. The second part investigated the use of virtual and 3D printed spine models for the surgical planning of complex spine deformity correction. Compared to usual radiographic images, the printed model allowed optimal surgical intervention, reduced surgical time and provided better surgeon-patient communication. The third part assessed the use of polyetheretherketone rods auxiliary to titanium rods to reduce the stiffness of posterior spine fusion constructs. Using a finite element model of the thoracolumbar spine, the rods system showed a decrease in the overall stress of the uppermost instrumented vertebra when compared to regular fixation approaches. Finally, a retrospective biomechanical assessment of a lumbopelvic reconstruction technique was investigated to assess the patients' gait following the surgery, the implant deformation over the years and the extent of bony fusion between spine and implant. In conclusion, this thesis highlighted the need to provide surgeons with new planning and assessment techniques to better understand postsurgical complications. The methodologies investigated in this project can be used in the future to establish a patient-specific planning protocol.

“Clinical and biological role of adjuvant dendritic cells vaccination in newly glioblastoma patients”

Background. Glioblastoma (GBM) is the most common primary tumor of central nervous system and it has a poor prognosis. Standard first line treatment, which includes surgery followed by adjuvant radio-chemotherapy,produces only modest benefits to survival. The interest for immunotherapy in this field derives from the development of new drugs and effective therapies as immune-check points inhibitors, adoptive T-cell approaches or dendritic cell (DC) based vaccines or a combinations of these. GBM is described as a typical “immune-deserted” cancer exhibiting a number of systemic and environmental immunosuppressive factors. Considering the role of microenvironment, and above all the lower tumor load and depletion of immunosuppressive cells in GBM, our hypothesis is that DC vaccine may induce an immune response. Main aims and study design. The main aim of this project is to study the role of immune system in GBM, including identification of potential prognostic and predictive markers of outcome and response to dendritic cell vaccine. Firstly, we performed a retrospective analysis on blood samples. Then, we analyzed the immuno-component in tissues samples of enrolled patients; and compared that with blood results. Then, the last part of the project is based on a prospective clinical trial on patients enrolled in DC-based vaccination produced at IRST Cell Factory and actually used for patients with melanoma and other tumors. The enrollment is still ongoing. Expected results. The project will i) develop an immune-panel of prognostic and predictive markers to help clinicians to improve the therapeutic strategy for GBM patients; ii) provide preliminary results on the effectiveness of immunotherapy on GBM patients.

Numerical simulations of the spine for patient-specific models of severe scoliosis

In silico methods, such as musculoskeletal modelling, may aid the selection of the optimal surgical treatment for highly complex pathologies such as scoliosis. Many musculoskeletal models use a generic, simplified representation of the intervertebral joints, which are fundamental to the flexibility of the spine. Therefore, to model and simulate the spine, a suitable representation of the intervertebral joint is crucial. The aim of this PhD was to characterise specimen-specific models of the intervertebral joint for multi-body models from experimental datasets. First, the project investigated the characterisation of a specimen-specific lumped parameter model of the intervertebral joint from an experimental dataset of a four-vertebra lumbar spine segment. Specimen-specific stiffnesses were determined with an optimisation method. The sensitivity of the parameters to the joint pose was investigate. Results showed the stiffnesses and predicted motions were highly depended on both the joint pose. Following the first study, the method was reapplied to another dataset that included six complete lumbar spine segments under three different loading conditions. Specimen-specific uniform stiffnesses across joint levels and level-dependent stiffnesses were calculated by optimisation. Specimen-specific stiffness show high inter-specimen variability and were also specific to the loading condition. Level-dependent stiffnesses are necessary for accurate kinematic predictions and should be determined independently of one another. Secondly, a framework to create subject-specific musculoskeletal models of individuals with severe scoliosis was developed. This resulted in a robust codified pipeline for creating subject-specific, severely scoliotic spine models from CT data. In conclusion, this thesis showed that specimen-specific intervertebral joint stiffnesses were highly sensitive to joint pose definition and the importance of level-dependent optimisation. Further, an open-source codified pipeline to create patient-specific scoliotic spine models from CT data was released. These studies and this pipeline can facilitate the specimen-specific characterisation of the scoliotic intervertebral joint and its incorporation into scoliotic musculoskeletal spine models.