Protective and regenerative therapies of the CNS

Fernando Pitossi - Fundación Instituto Leloir

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The general aim of our laboratory is to provide information to design protective or regenerative therapies for Parkinson´s Disease (PD).  Unfortunately, there is no current therapy that modifies disease progression. The death of the dopaminergic neurons of the nigrostriatal system is the primary cause of the motor symptoms observed in these patients. Microglial activation in the nigrostriatal system is ubiquitously found in animal models of PD and PD patients. Our laboratory has been focusing on the study of neuroinflammation in PD during the last 17 years to identify new therapeutic targets that could be the basis of protective therapies against this disease. The first question we wanted to ask was: is microglial activation only a consequence of neuronal death or it has functional effects on neuronal survival and therefore could be regarded as a therapeutic target? At the beginning, we have observed that microglial activation possesses toxic as well as neuroprotective effects during dopaminergic degeneration and that the cytokines secreted by them are instrumental to these effects. Therefore, our second question was: which factors determine univocal effects (toxic or neuroprotective) of brain cytokines during neurodegeneration? Searching for this answer, we have generated 5 animal models of neurodegeneration by the modulation of neuroinflammation by cytokines. In these models, the magnitude, duration and timing of cytokine expression allow to predict a univocal effect, enabling in depth studies on the mechanism of action implicated. We hypothesize that mediators exist that are common to the toxic effects of cytokines in these 5 models generated by different pro-inflammatory stimuli. We pretend to identify those common molecules downstream of the inflammatory stimuli by functional genomics in the models generated; becoming independent from the factors that alter the final functional effect of the cytokines. On the other hand, since 2000 we study adult neural stem cells (NSC) as a putative source for regenerative therapies for PD. In particular, we study the effects of cytokines on the proliferation, differentiation and survival of NSC endogenously and after transplantation in the brain. We have observed a pro-neurogenic effect of TGF-beta 1, an anti-inflammatory cytokine, on NSC in vivo and in vitro in 3 different experimental models. Based on these and other results, the field has incorporated cytokines as part of the neurogenic niche. We are now focusing in studying the mechanism of action of TGF-beta1 in vivo and in vitro by functional genomics. In addition, we have developed the technology of cellular reprogramming as a source of dopaminergic neurons from fibroblasts for future regenerative therapies and as in vitro models of PD. This technology will allow merging all the information and animal and cellular models that we have generated. On one hand, the therapeutic targets for protective therapies that we identify will be tested on dopaminergic neurons derived from iPS cells from PD patients. On the other, every cell transplantation in the brain will elicit inflammation. Therefore, the information on the functional effects of cytokines on cell proliferation, differentiation and survival will be investigated on dopaminergic neurons transplanted into traditional PD models or PD models based on neuroinflammation generated in our laboratory.  We are currently members of a consortium with the California Institute for Regenerative Medicine and the NIH to design and implement a clinical trial against PD using dopaminerig neurons derived from pluripotent cells.
Aim 1. Identify molecular mediators of the toxic effects of neuroinflammation in PD
As an effort to develop neuroprotective therapies against PD, we seek to identify inflammation-related molecules that could be defined as therapeutic targets. We hypothesize that there are common mediators to the toxic effects of inflammation in the nigrostriatal system. We are applying functional genomics on 5 different animal models where inflammation triggered by several inflammatory stimuli generates nigrostriatal neurodegeneration and motor symptoms (for more detailed information on these models, refer to: Neurobiology of Disease, 2006 Oct;24(1):183-93; Brain, 2008, 131:1880-94, Neurobiology of Disease, 2010, 37(3):630-40 J Neuroimmunol. 2010, May;222(1-2):29-39 and Exp Neurol. 2011 Feb;227(2):237-51). The toxic effects of the identified genes will be tested on dopaminergic neurons derived from iPS cells and animal models of PD.

Aim 2. Identify the molecular mechanism of the pro-neurogenic effect of TGF-beta1
We have identified and validated differential genes that are candidates to mediate the pro-neurogenic effects of TGF-beta1 by functional genomics. We plan to incorporate this information on the differentiation protocol of neuronal precursors derived from iPS cells already generated in our laboratory.

Aim 3. Regenerative therapies: pre-clinical studies
We seek to test the safety and efficacy of the transplantation of dopaminergic neurons derived from iPS cells on animal models of PD. We also plan to improve the safety and efficacy of the protocol by including anti-inflammatory treatments and/or expression of genes related to inflammation.

Wenker SD, Pitossi FJ. Cell therapy for Parkinson's disease is coming of age: current challenges and future prospects with a focus on immunomodulation. Review. Gene Ther. 2019 Apr 16. doi: 10.1038/s41434-019-0077-4

Galeano P, Leal MC, Ferrari CC, Dalmasso MC, Martino Adami PV, Farías MI, Casabona JC, Puntel M, Do Carmo S, Smal C, Arán M, Castaño EM, Pitossi FJ, Cuello AC, Morelli L. Chronic Hippocampal Expression of Notch Intracellular Domain Induces Vascular Thickening, Reduces Glucose Availability, and Exacerbates Spatial Memory Deficits in a Rat Model of Early Alzheimer. Mol Neurobiol. 2018 Nov;55(11):8637-8650.

Silva BA, Leal MC, Farías MI, Avalos JC, Besada CH, Pitossi FJ, Ferrari CC. A new focal model resembling features of cortical pathology of the progressive forms of multiple sclerosis: Influence of innate immunity. Brain Behav Immun. 2018 Mar;69:515-531

Rosato-Siri MV, Marziali L, Guitart ME, Badaracco ME, Puntel M, Pitossi F, Correale J, Pasquini JM. Iron Availability Compromises Not Only Oligodendrocytes But Also Astrocytes and Microglial Cells. Mol Neurobiol. 2017 Jan 14. doi: 10.1007/s12035-016-0369-2

Miraglia MC, Costa Franco MM, Rodriguez AM, Bellozi PM, Ferrari CC, Farias MI, Dennis VA, Barrionuevo P, de Oliveira AC, Pitossi F, Kim KS, Delpino MV, Oliveira SC, Giambartolomei GH. Glial Cell-Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome-Dependent IL-1β Production. J Immunol. 2016 May 1;196(9):3794-805.

Wenker SD, Leal MC, Farías MI, Zeng X, Pitossi FJ. Cell therapy for Parkinson׳s disease: Functional role of the host immune response on survival and differentiation of dopaminergic neuroblasts. Brain Res. 2016 May 1;1638(Pt A):15-29. Review

Wenker SD, Casalía M, Candedo VC, Casabona JC, Pitossi FJ. Cell reprogramming and neuronal differentiation applied to neurodegenerative diseases: Focus on Parkinson's disease. FEBS Lett. 2015 Nov 14;589(22):3396-406. Review

Palma V, Pitossi FJ, Rehen SK, Touriño C, Velasco I. Stem cell research in Latin America: update, challenges and opportunities in a priority research area. Regen Med. 2015;10(6):785-98. Review  

Radice PD, Mathieu P, Leal MC, Farías MI, Ferrari C, Puntel M, Salibe M, Chernomoretz A, Pitossi FJ. Fibulin-2 is a key mediator of the pro-neurogenic effect of TGF-beta1 on adult neural stem cells. Mol Cell Neurosci. 2015 Jun 5;67:75-83

Murta V, Farías MI, Pitossi FJ, Ferrari CC. Chronic systemic IL-1β exacerbates central neuroinflammation independently of the blood-brain barrier integrity. J Neuroimmunol. 2015 Jan 15;278:30-43

Solomon S, Pitossi F, Rao MS. Banking on iPSC--is it doable and is it worthwhile. Stem Cell Rev. 2015 Feb;11(1):1-10. Review

Pitossi FJ, Podhajcer OL. Current status of stem cells and regenerative medicine research in Argentina. Stem Cells Dev. 2014 Dec;23 Suppl 1:17-9. Review





Fernando Pitossi
Head of Laboratory - fpitossi@leloir.org.ar



María Celeste Leal
Assistant Research - CONICET



Berenice Silva
PhD Student - Fundación René Baron



Brenda Erhardt
Ph.D. Student - CONICET



Esteban Miglietta
Ph.D. Student - CONICET



María Isabel Farias
Technical assistant



Victoria Gradaschi
Passant