The main focus of our group is to investigate the molecular mechanisms that cause neurodegenerative pathologies, mainly Parkinson’s disease (PD) and Alzheimer’s disease (AD), in order to exploit this knowledge for drug development and technological applications of amyloid aggregates.

Drug repurposing and development:

The increased longevity of the human population situates neurodegenerative diseases as a critical challenge to health care systems throughout developed countries. In fact, according to a systematic study on PD, approximately 4.6 million Western European patients were diagnosed in 2005, and that number is expected to increase to approximately 9 million in 2030. These data highlight the urgent need for identification of effective neuroprotective therapies in order to avoid the collapse of the healthcare systems. In this scenario, treatments must directly target underlying disease pathogenesis. Sinceabnormal aggregation of specific proteins like a-synuclein, tau, prionic protein or TDP-43, among others, would seem to be the common cause foralmost all neurodegenerative diseases. Then, inhibiting this anomalous aggregation or promoting its removeare nowadaysmain target of drug design. Unfortunately, despite the significant investment in research and drug development, to date, all attempts have failed.

 Oxidative stress, mitochondrial dysfunction, and neuro-inflammation have also been recognized as triggers of neuronal cell death in neurodegenerative disorders. As an example,intertwining relationship between the above elicits, compelling evidence shows that α-synucleinaggregation can trigger all these events, and sometimes vice versa, suggesting that these processes would foster a vicious cycle with fatal consequences for neurons.

Despite significant number of molecules recognized as efficient in vitro inhibitors of α-synuclein amyloid self-assembly they have failed to provide an effective neuro-protection in vivo. In addition, drugs developed to block these processes individually proved ineffective in clinical trials. Therefore, the big challenge today must be centered in finding molecules that target these multiple processes simultaneously in order to reach an efficient neuroprotective effect. That is, we must think of a multitask drug, capable of inhibiting aggregation, neuroinflammation and being also a good antioxidant molecule to prevent the mitochondrial damage.

In this context,our studies in Doxycycline as a potential neuroprotector aquiere relevance. In fact, thissecond-generation tetracycline, can attenuate the pathological cycle induced by aggregated species of α-synuclein (described above) at five different points: inhibiting α-synuclein aggregation and seeding, neuro-inflammation in glia cells, mitochondrial dysfunction and ROS damage. Besides its neuroprotective effect and safety as benefits, the lipophilicity of doxycycline allows good penetration (26%) through the blood brain barrier (BBB).

 A) Repurposing Doxycycline:

In cells, proteins achieve their function by folding into 3D identities. Only if they undertake their unique and correct shape, can they perform their roles that support life. Neurodegenerative disorders are caused by aberrant auto-association of specific proteins, which becomes toxic to brain cells. The protein related with this abnormal aggregation en PD is called a-synuclein while tau and amyloid-β (Aβ) with AD. In fact, under specific environment conditions they have a tendency to clump together forming nanofibrils.

We have recently shown that doxycycline, and safe, well-know, and widely prescribed antibiotic, can interfere with the pathologic cycle involved in a-synuclein abnormal aggregation. In fact, we have demonstrated for the first time that doxycycline can interact with aggregated toxic species of alpha synuclein, remodeling them into a less toxic version of the protein. This ultimately leads to enhanced survival in cell culture assays.

We have calculated, by extrapolating our experimental data, that doxycycline in sub-antibiotic doses (20–40 mg/day) would be sufficient for neuroprotection. For this reason, we propose that doxycycline, in subantibiotic doses, could be repurposed as neuroprotectorfor Parkinson’s disease.

Considering that doxycycline has been used worldwide for 50 years, its safety, bioavailability, metabolism, toxicity and ability to cross the BBB are well known. This valuable knowledge allows to avoid preclinical trials with the concomitant save of time and money. Then, this multitask drug is waiting the Phase I clinical trial as neuroprotector in newly diagnosed PD, ie with a well selected group of patient with low UPDRS to check if it is able to slow down or stop the neurodegenerative process.

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 B) Drug development

The main objective of this project is the designnovel moleculefamilies based on our knowledge in structure-function relationship and structural predictions from tetracyclines and other molecules studied in our lab. We havethe structural determinants for the atiaggregant properties definedand thus we are working in collaboration with chemical synthesis experts in order to obtain different compounds families. To do this, we are working with the collaboration of SkyBio LLC and two prestigious chemist groups from University of Buenos Aires (UBA) under Dr. Oscar Varela advise, and whit Dr. Bruno Figader advise at Université de Paris Sud.

Our main aim is the developing novel molecules with increased neuroprotective efficiency, but less antibiotic activity, while preserving their ability to penetrate the blood brain barrier. The ability to reach good concentrations in the CNS is essential in order to remodel toxic species of a-synuclein and tau that could slow down neurodegenerative processes.

Novel diagnostic methods for Neurodegenerative diseases:

 Aggregates species of proteins like a-synuclein, tau, prionic protein and TDP-43, among others, are nowadays considered responsible for neuronal death in neurodegenerative diseases like Parkinson’s and Alzheimer’s. Unfortunately, to date, there is no reliable biomarker to detect, nor follow, the evolution of these pathologies. Quantification of aggregated species of these proteins in biological fluids would represent the most promising strategy for early detection of these disorders. In these cases, classical techniques such as ELISA do not possess the sensitivity, reliability nor reproducibility necessary to be a wide spread diagnostic and prognostic method. This is due to the great difficulty of obtaining specific antibodies to recognize the great diversity of aggregated species.

Currently, there is no biochemical blood or cerebrospinal fluid (CSF) analysis or diagnostic test for PD, and clinical manifestations in early stages are difficult to detect. The lack of awidespread analysis or diagnostic test for PD and AD is relevant if we consider that novel therapies against neurodegenerative disorders will require the ability to detect their earliest possible presymptomatic manifestations, since lost neurons generally cannot be replaced.

In this context, the main goal of this project is to develop and validate novel tools for early detection of a-synuclein and tau aggregates in biological fluidsand novel PET radiotracers based on our knowledge of the molecular determinants that strongly bind toxic species of a-synuclein and tau. This development would revolutionize the field of neurodegenerative pathologies.

Technological applications of amyloid aggregation

 This project aims to develop novel technology based on protein aggregation by harnessing our experience in biophysical characterization of proteins and formation of amyloid nanofibers. For example, one technological platform aspires to use amyloid structures as support for the immobilizing molecules of technological interest in the design of biosensors and biocatalysts.

Autophagy and lysosomal biology in neurodegenerative diseases:

The protein that forms toxic aggregates in Parkinson’s disease, a-synuclein, is normally degraded through the lysosomal-autophagy system. Mutations that affect this system can alter a-synuclein levels, leading to increased amyloid aggregation. Previous mechanistic studies have uncovered the relationship between lysosomal dysfunction and the toxic a-synuclein aggregates that lead to neurodegeneration.  Although a-synuclein is an abundant protein, even subtle elevations in its levels are capable of driving pathological aggregation, since this polymerization event is highly concentration-dependent.

Although most forms of neurodegenerative diseases like Parkinson’s are thought to be idiopathic, genetic research have pointed to a clear association with specific lysosomal gene variants as risk factors. Many risk-associated genes converge on cellular clearance pathways involving lysosomes and autophagy.

A new research line within IMMCA will be dedicated to investigating how regulation of lysosomal and autophagosomal biogénesis and function affects a-synuclein-induced neurodegeneration. In this context, this project aims at understanding the curious relationship between Parkinson, melanoma and a specific family of transcription factors. Understanding how to boost degradation pathways within specific neurons in order to degrade toxic accumulation of protein aggregates can ultimately lead to novel therapies and prevention of neurodegenerative diseases.

High quality heterologous production of neurodegenerative research-relevant proteins – SkyBio

 The main objective of this project is to collaborate with the SkyBio (link) in order to produce heterologous recombinant proteins and peptides of high quality and purity with a focus on neurodegenerative disease research and diagnostic. Our interest is to apply and improve our know-how in a-synuclein, tau, and GAPDH protein production as well as commercialization strategies. Moreover, we aim to produce and purify these proteins with post-translational modifications like phosphorylation, acetylation, and aggregation, which are strongly required for basic research and novel bio-diagnostic kits.