Prelegenci 

Beta-propeller Protein-Associated Neurodegeneration (BPAN), a subtype of the rare group of “Neurodegeneration with Brain Iron Accumulation” (NBIA) disorders, displays unique pathological hallmarks including iron build-up in the substantia nigra, prominent degeneration of nigral dopamine neurons, and early-onset parkinsonism. However, the molecular mechanisms contributing to the distinctive loss of nigral dopaminergic neurons, and what role iron dysregulation may play in this, remain enigmatic. To model the unique pathology in this neuronal subtype, we differentiated BPAN patient-derived induced pluripotent stem cells (iPSCs) into long-term cultures of midbrain dopaminergic neurons. Our model system revealed altered protein expression in BPAN neurons, most notably in regulators of iron and dopamine homeostasis, and disrupted dopamine metabolism resulting in accumulation of oxidized dopamine. This oxidation is catalysed by iron and culminates in the formation of neuromelanin, a neuroprotective process that sequesters potentially toxic metals and reactive compounds. However, excess iron and/or reactive intermediates of dopamine oxidation may saturate the neuromelanin system, eventually contributing to neurotoxicity. Of note, iron dyshomeostasis and excessive oxidized dopamine has been demonstrated in post-mortem tissue and iPSC-derived neurons from various Parkinson’s disease (PD) patients. Thus, findings from our work may represent shared pathological mechanisms between BPAN and the second most common neurodegenerative disease, PD. Future explorations into the intersection of these pathways may reveal promising new strategies for targeted protection of the highly vulnerable nigral dopaminergic neurons, potentially impacting patient populations well beyond BPAN and NBIA.

One of the subtypes of NBIA is BPAN, which is caused by a mutation in the WDR45 gene. Since the genetic defect in BPAN, consisting in a mutation in the above gene, results in disorders of the autophagy process, it seems natural that stimulation of an inefficient cellular process may bring a significant improvement in cell functioning, and thus, therapeutic benefits expressed in the improvement of the clinical condition of patients. Genistein [5,7- dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is one of the flavonoids belonging to the group of isoflavones, exhibiting a wide spectrum of biological activities. This molecule has the ability to induce many molecular effects that can be used for therapeutic purposes, such as antioxidant activity, inhibition of inflammation, promotion of apoptosis, modulation of the activity of steroid hormone receptors and metabolic pathways. In addition to the above- mentioned features of genistein, it is characterized by additional aspects of action that make it a very good candidate for a drug for neurodegenerative diseases. First of all, literature data indicate the inactivation of mTOR kinase in various types of cells under its influence, which is a signal to inhibit the processes of protein synthesis and induction of autophagy. The aim of the project was to use genistein in pediatric patients with BPAN, under the care of the Pediatric Neurology Department of the Medical University of Lublin. Six-month observations indicate clinical improvement, especially in mildly symptomatic patients. Positive changes were observed primarily in the cognitive but also motor sphere. Work is currently underway on the use of genistein in other varieties of NBIA.

Genetic alterations within the C19orf12 gene lead to the development of Mitochondrial Membrane Protein Associated Neurodegeneration (MPAN), a rare hereditary neurological disorder included in the Neurodegeneration with Brain Iron Accumulation category (NBIA4). C19orf12 codes for a small protein located in the mitochondria, endoplasmic reticulum, and at sites associated with mitochondrial membranes. The function of the protein as well as the molecular underpinnings of the disease are poorly described. Experimental results point to an involvement of the protein in lipid metabolism, maintenance of mitochondrial function, and the regulation of autophagy. We exploited the zebrafish experimental system to investigate C19orf12 biology. There are four counterparts of the human gene in the zebrafish genome: c19orf12a located on chromosome 18, and c19orf12b1, b2, and b3, clustered in tandem on chromosome 7. According to available RNA-seq data c19orf12a is expressed at higher levels during the early stages of zebrafish development. The WISH analysis showed a similar expression pattern in CNS and somites for all the paralogues at 24 hpf.A zebrafish morphant with a knockdown of the c19orf12a gene displayed abnormal brain morphology, a smaller head and eyes, reduced yolk extension, a tilted and thinner tail, and a significant disruption in musculature formation, leading to impaired locomotor behavior. The co-injection of the human C19orf12wild-type mRNA restored the normal morphology, whereas the co-injection of the mutant mRNA failed to do so.To delve deeper into the mechanisms behind neurodegeneration and the late-onset effects in adult zebrafish, we developed stable c19orf12 loss-of-function models using the genome editing technology. So far, we have generated two stable c19orf12amutant lines: one with a 2 bp deletion (Δ2) leading to a premature stop codon, and another with an in-frame, potentially pathogenic,3 bp deletion (Δ3). We are performing a comprehensivephenotypic evaluation including the assessment of neuronal development and behavior. The initial characterization did not reveal any significant morphological alterations, although the Δ3 F4 generation, not provided with wild-type maternal mRNA, exhibited a significant larval lethality. The mutants show perturbation in the locomotor behavior, such as an increase in the frequency of the tail flipping at 24 hpf and a longer distance swamat the end of a cycle of alternating light–dark periods at 120 hpf. Further analysis is ongoing to investigate the lipid metabolism and the autophagy flux in mutant embryos and larvae.  

Gene therapy is an emerging precision therapy for a number of rare early-onset neurogenetic disorders, with licensed therapies already available for spinal muscular atrophy and AADC deficiency. Gene replacement therapy has the potential to be an efficacious therapy for NBIA disorders where the causative gene is known, given disease is usually attributed to a loss-of-function mechanism. In this lecture, I will present the status of gene therapy development for NBIA, with a focus on Infantile Neuroaxonal Dystrophy. I will discuss how common principles can be applied more broadly to the NBIA field.

To clarify the relationship between brain iron dys-homeostasis and neurodegeneration, we developed hiPS-derived astrocytes of PANK2- and COASY-associated neurodegeneration (PKAN and CoPAN). They are two NBIAs caused by mutations in genes that codify for key enzymes in Coenzyme A biosynthetic chain reactions. Both diseases are characterized by progressive neurodegeneration and a huge iron-accumulation in the globus pallidus of patients. PKAN astrocytes showed cytosolic iron accumulation, alteration of iron metabolism, mitochondria morphology, respiratory activity, oxidative status, signs of ferroptosis and were prone to develop a stellate-like phenotype, thus gaining a neurotoxic feature, as confirmed by co-cultures of PKAN astrocytes and glutamatergic neurons. Experiments ran in CoPAN astrocytes showed cytosolic iron overload, a tendency to acquire a stellation phenotype and a positive significant correlation between the stellation grade and the amount of up-taken transferrin, suggesting a potential impairment in membrane dynamics that could be at the basis of iron overload also in CoPAN astrocytes. Analysis of constitutive exo-endocytosis, a key route for cellular iron intake, and vesicular dynamics, by exploiting the activity-enriching biosensor SynaptoZip, led to the finding of a general impairment in the constitutive endosomal trafficking in PKAN astrocytes. Super-resolution (SRFF) experiments, confirmed an impaired intracellular fate of transferrin-loaded endosomes. In addition, cytosolic and mitochondrial aconitase activity, heme content, as well as the mitochondrial redox-active labile iron pool resulted strongly reduced respect with the controls. Thus, the impairment of iron delivery to mitochondria appears the cause of cell iron delocalization, resulting in cytosolic iron overload and mitochondrial iron deficiency, that triggers mitochondria dysfunction promoting neurodegeneration. These astrocyte models result helpful not only to clarify pathogenetic mechanisms that lead to iron overload, but also to find common routes in two exemplar NBIA disorders. Furthermore, they allowed to test new therapeutic options, as for example the PPAR Gamma Agonist Leriglitazone that we found to be efficient in ameliorating mitochondrial function in PKAN astrocytes.

The WDR45 gene, which when mutated causes BPAN, produces a protein called WDR45 that is also involved in autophagy, the process in which the body’s cells eliminate damaged or unnecessary components. The precise molecular function and contribution of this protein to this pathway, however, remains unclear. Depletion or mutations in WDR45 are also causing dysregulation in iron metabolism and endoplasmic reticulum morphology. Furthermore, we and others have shown that mitochondria, which are the small organelles in cells responsible for important processes such as energy production, metabolism regulation, inflammation and cell death, are affected by the inactivation of the WDR45 protein.

Although genes affected in different types of neurodegeneration with brain iron accumulation (NBIA) have been identified, molecular mechanisms of these diseases are not yet fully understood. On the other hand, only knowing details of pathomechanism of a specific disease may allow to develop effective therapies. Since NBIA is characterized by iron accumulation, the process of ferroptosis (programmed cell death dependent on iron) has been suspected to be involved in the pathogenesis of this disease. Preliminary experiments indicated that expression of some genes coding for proteins involved in ferroptosis may be dysregulated in various NBIA diseases. This discovery, accompanied with studies on levels of specific ferroptosis-related proteins, opened a new field in the studies on NBIA which may lead to identification of as yet unknown mechanisms of this disease and can facilitate studies on development of novel therapeutical strategies.

The role of the protein encoded by C19orf12 remains unknown, but it appears to be related to mitochondrial function.Autopsy studies of patients with NBIA-MPAN showed similar neuropathological changes that have been described in Parkinson's disease (PD). Mitochondrial dysfunction is a key element in the development of the PD. Lipid peroxidation causes α-synuclein-induced neurotoxicity. The role of omega-3 PUFAs in adjuvant therapy in PD has been investigated in both experimental models and clinical trials. It was found that DHA used in the mouse model prevented the loss of substantia nigra cells. In clinical trials, omega 3 PUFA resulted in improvement of both neurological performance, disease severity and mood disorders during 3-6 months of use. Omega-3 PUFA supplementation is associated with improved cognitive functions - it increases the number of synapses, stimulates the growth of nerve cells, and increases the concentration of the BDNF factor Additionally, it has been proven that the use omega 6 - linoleic acid or C19orf12 protein in the PLAN animal model rescued neuronal phenotype.We designed a clinical study for MPAN patients with omega 3 and 6 PUFA. Results: Patients were treated for 6 months with omega 3,6 – randomized for 2 different doses. They were assessed at the entering the study and after 6 months. We have recorded changes in neurological status and biomarker status in all patients.  

The title of her presentation is “Physiotherapy assessment of disease progression” She will talk about changes occurring in the gross and fine motor skills in patients with NBIA and discuss physiotherapy approaches.

One of putative treatment options in various genetic and neurodegeneration diseases which are caused by accumulation of macromolecular aggregates in cells is stimulation of the autophagy process. Such a therapeutic strategy is based on the assumption that induced autophagy might be helpful in removal of the toxic aggregates from cells, thus resuming the balance in the cell physiology. The problem is, however, that overstimulation of the autophagy process may lead to cell destruction. Already known autophagy stimulators are very effective in vitro, however, they can cause severe adverse effects in patients, especially when used for a log time. It is therefore proposed to stimulate autophagy using a natural compound, a molecule from the group of flavonoids, which can induce autophagy efficiently, while gently enough to avoid auto-destruction of cells and adverse effects. Studies with cellular models suggested that such a strategy might be effective in different diseases, including at least some types of neurodegeneration with brain iron accumulation (NBIA).