Proteinopathies: from mechanisms to therapies

Stuctural diversity of misfolded proteins

In most cells of all organisms, the most diverse proteins are constantly produced during protein synthesis, which perform a wide variety of functions in the cell and throughout the organism. For a protein to function properly, its tertiary structure is of crucial importance. This structure is achieved through a process called protein folding.

Abnormal proteins in certain proteinopathies can fold into different three-dimensional shapes; these variant protein structures are defined by their different pathogenic, biochemical and conformational properties. In these diseases, the proteins amyloid structure is composed of the cross-β-sheet entity, which is an almost indefinitely repeating two-layered intermolecular β-sheet motif. The misfolded proteins can become toxic (a toxic gain-of-function) or can lose their normal function. Protein misfolding also increases the tendency of specific proteins to bind to themselves.  These aggregated proteins are able to propagate and multiply in the brain, causing the spreading of the pathology in the brain.

Proteinopathies include Alzheimer or Parkinson’s diseases or amyloid peripheral neuropathy, and a wide range of other disorders. Our teams evaluate mechanisms associated to proteionopathies and investigate potential therapies to prevent or cure these diseases. They focus on three pathologies: i. Peripheral neuropathy caused by amyloidogenic transthyretin (ATTRv); ii. Diseases associated with beta-amyloid and tau proteins (Alzheimer disease, fronto-temporal dementia); iii. Diseases associated with alpha-synuclein (Parkinson’s disease, Lewy body disease).

Our research unit investigate

– Interactions between misfolded proteins and synapses

– Regulation of misfolded protein synthesis and clearance towards identification of therapeutic targets

– Therapies to cure misfolded proteins with the first ever efficient anti-amyloid therapy conducted in peripheral neuropathy.

Interaction between misfolded proteins and synapses

Impact of α-synuclein dynamics at synapses and axons

PI: Christian Specht (Team 1)

Parkinson’s disease or Lewy body disease are caused by alpha-synuclein that form Lewy bodies (LB) and induce neurodegeneration. Toxic oligomers and fibrillar forms of α-synuclein trigger early pathological changes at synapses and throughout the neuron. Despite detailed in vitro biochemical analysis, there is no direct experimental evidence for the earliest stages of α-synuclein dysregulation in the neurons that cause synucleinopathies. C. Specht team uses single molecule localization microscopy (SMLM) to visualize and quantify early processes of α-synuclein toxicity at synapses and axons. SMLM is ideal for exploring these processes due to recent technical advances. Quantitative super-resolution imaging allows analysis of precise spatial relationships between synaptic components and determination of copy number with single-molecule sensitivity. α-Synuclein mobility is measured by SMLM in living neurons to extract diffusion properties and its trapping at synapses, both under control and pathogenic conditions. By combining single molecule diffusion with statistical data analysis, we can measure changes in the strength of α-synuclein interactions at synapses, evaluate the interaction of α-synuclein with different markers and assess how different therapies can modulate  α-synuclein toxicity.

Verdier H, Laurent F, Cassé A, Vestergaard CL, Specht CG*, Masson JB* (2023). Simulation-based inference for non-parametric statistical comparison of biomolecule dynamics. PLoS Comput Biol 19:e1010088.

Specht CG (2021). A quantitative perspective of alpha-synuclein dynamics – why numbers matter. Front Synaptic Neurosci 13:753462,

Synaptic activity and protection against misfolded tau protein 

PI: Yvette Akwa (Team 1), Davide Tampellini (Baulieu Institute, Team 1)

Changes in synaptic excitability and reduced brain metabolism are among the earliest detectable alterations associated with the development of Alzheimer’s disease (AD). Stimulation of synaptic activity has been shown to be protective in models of AD beta-amyloidosis. Remarkably, deep brain stimulation (DBS) provides beneficial effects in AD patients, and represents an important therapeutic approach against AD and other forms of dementia.

Our group investigated the effect of synaptic stimulation on Tau pathology and synapses in in vivo and in vitro models of AD and frontotemporal dementia (FTD). Chronic DBS or chemically induced synaptic stimulation reduced accumulation of pathological forms of Tau and protected synapses, while chronic inhibition of synaptic activity worsened Tau pathology and caused detrimental effects on pre- and post-synaptic markers, suggesting that synapses are affected.

Interestingly, degradation via the proteasomal system was not involved in the reduction of pathological Tau during stimulation. In contrast, chronic synaptic activation promoted clearance of Tau oligomers by autophagosomes and lysosomes. Chronic inhibition of synaptic activity resulted in opposite outcomes, with build-up of Tau oligomers in enlarged auto-lysosomes. In addition, the stimulation of synaptic activity activates the transcriptor factor EB (TFEB) a master regulator of lysosomal function and autophagy, and promotes the clearance of pathological tau oligomers in P301S mutant tau cells. These data indicate that synaptic activity counteracts the negative effects of Tau in AD and FTD by acting on autophagy, providing a rationale for therapeutic use of DBS and synaptic stimulation in tauopathies.

Akwa Y, Gondard E, Mann A, Capetillo-Zarate E, Alberdi E, Matute C, Marty S, Vaccari T, Lozano AM, Baulieu EE, Tampellini D. (2018) Synaptic activity protects against AD and FTD-like pathology via autophagic-lysosomal degradation. Mol Psychiatry. 23:1530-1540.

Akwa Y, Di Malta C, Zallo F, Gondard E, Lunati A, Diaz-de-Grenu LZ, Zampelli A, Boiret A, Santamaria S, Martinez-Preciado M, Cortese K, Kordower JH, Matute C, Lozano AM, Capetillo-Zarate E, Vaccari T, Settembre C, Baulieu EE, Tampellini D. Stimulation of synaptic activity promotes TFEB-mediated clearance of pathologic MAPT/Tau in Cellular and mouse models of tauopathies. Autophagy 2023, 19: 660-677

Regulation of signaling transduction by unnatural amino acids

PI : Shixin Ye-Lehman (Team 1)

Dr. Shiwin Ye-Lehman, is developing synthetic biology tools to address fundamental problems associated with signaling transduction mechanisms in neuronal diseases. Synthetic biology is an interdisciplinary branch of biology and engineering, which enables to dissect the precise synthesis of biomolecules in vivo. Dr Ye-Lehman has developed efficient methods to genetically encode unnatural amino acids (UAA) into membrane proteins (including G protein-coupled receptors, ligand-gated ion channels) to study their structure-function relationships. She demonstrated the feasibility to expand the genetic code in mammalian cells, Xenopus laevis oocytes, primary neurons, and recently two animal models (mice and zebrafish). The incorporation of light-sensitive UAA has elucidated a novel molecular mechanism that explains functional differences between N-methyl-D-aspartate (NMDA) receptor subtypes, which play key roles in the excitatory synaptic transmission associated with learning and memory. In the course of these studies, she has identified series of light-sensitive NMDA receptors whose activities can be modulated by light. Currently she is developing new optical methods to regulate neuronal signaling processes with light, and we are engineering light-responsive neuronal receptors for optogenetic studies. These tools will be used to regulate synaptic impairments associated with proteinopathies.

GluN2A and GluN2B NMDA receptors use distinct allosteric routes. Tian M, Stroebel D, Piot L, David M, Ye S*, Paoletti P*. Nat Commun. 2021 Aug 5;12(1):4709.

Regulation of misfolded protein synthesis and clearance towards identification of therapeutic targets

Protection from tau pathology through TSPO-related regulation of steroid synthesis 

PI: Yvette Akwa (Team 1), Davide Tempellini (Baulieu Institute, Team 1), Michael Schumacher (Team 1)

The progressive hyperphosphorylation and aggregation of tau proteins in the brain forms aberrant filamentous inclusions giving rise to neurofibrillary tangles (NFT), the feature of tauopathies. The presence of tau aggregates is associated with synaptic loss, mitochondrial impairments and neuroinflammation.  The 18 kDa translocator protein (TSPO/PBR) is a mitochondrial membrane protein that participates in steroidogenesis. Many studies including ours showed that TSPO ligands stimulate the endogenous production of neuroactive steroids in mice and rats.  Additionally, besides its implication in neuroinflammation, published data indicate increased neuronal TSPO expression upon stimulation of neuronal activity. We hypothesized that TSPO ligands XBD173 and etifoxine treatment ameliorate tau hyperphosphorylation, tau oligomer clearance and impaired mitochondrial structure/activity in P301L cells and P301S mouse models of FTDP-17, by involving the stimulation of neurosteroidogenesis and/or TFEB activation. Complete deletion of tspo/pbr gene in P301L cells will show specific effects of TSPO ligands. Over the past decades, the use of genetic TSPO-knockout models has challenged the function of TSPO/PBR in steroid biosynthesis. We recently show that TSPO is not required for the novo synthesis of pregnenolone in the mouse adrenal glands and brain under basal conditions. A steroidome in wild-type TSPO-KO mice and TSPO-KO rats treated with etifoxine is under investigation. Overall, the modulation of TSPO/PBR activity could represent an attractive disease-modifying strategy in tauopathies.

Further studies are ongoing to evaluate the effects of direct administration of natural steroids or their synthetic enantiomers, particularly excitatory compounds stimulating neurotransmission.

El Chemali L, Akwa Y, Massaad-Massade L. The mitochondrial translocator protein (TSPO): a key multifunctional molecule in the nervous system. Biochem J. 2022, 479:1455-1466

Dementias and neurodegeneration : A role for FKBP52 in Tau function

PI : Étienne-Émile Baulieu, Julien Giustiniani (Baulieu Institute and Team 1)

FK506 Binding Protein 52 (FKBP52) is a protein that interact with the microtubule associated protein Tau. It is also involved in nervous system development (axonal growth, neuron viability). The team of Pr. Baulieu identified a direct interaction of FKBP52 with Tau-P301L and its phosphorylated forms. It demonstrated FKBP52’s ability to induce the formation of Tau-P301L oligomers. In a transgenic zebrafish expressing the human Tau-P301L mutant, FKBP52 knockdown is sufficient to redrive defective axonal outgrowth and branching related to Tau-P301L expression in spinal primary motoneurons. Thus, our group test the hypothesis that FKBP52 regultation can modulate Tau pathology evolution.

Chambraud B, Byrne C, Meduri G, Baulieu EE, Giustiniani J (2022) FKBP52 in neuronal Ssgnaling and neurodegenerative diseases: A microtubule story. Int J Mol Sci 23.

The first therapy to cure peripheral neuropathies induced by amyloidogenic transthyretin (ATTRv)

PI: David Adams (Team 2), Andoni Echaniz-Laguna (Team 2)

Hereditary amyloidogenic transthyretin (ATTRv) amyloidosis with polyneuropathy (also known as familial amyloid polyneuropathy) is a condition with adult onset caused by mutation of transthyretin (TTR) and characterized by extracellular deposition of amyloid and destruction of the somatic and autonomic peripheral nervous system, leading to loss of autonomy and death.  The pathophysiology of the neuropathy includes instability and proteolysis of mutant TTR leading to deposition of amyloid with variable lengths of fibrils, microangiopathy and involvement of Schwann cells. Wild- type TTR is amyloidogenic in older individuals. The main symptoms are neuropathic, but the disease is systemic leading to cardiac, eye and kidney pathology.

Until recently, disease modifying therapeutics that have been developed include liver transplantation and TTR stabilizers,
both of which can slow progression of the disease and increase survival in the early stages.

D. Adams (head of the Neurology Department of the Bicêtre Hospital and member of Team 2) has developped the first-ever siRNA therapeutic for the treatment ATTRv, in collaboration with Alnylam Pharmaceuticals (Adams et al., 2018). The siRNA product Patisiran (Onpattro) has been approved by American Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of hereditary transthyretin-mediated amyloidosis in adult patients after a successful Phase III trial.

Major problems faced by siRNA therapy are their rapid degradation and the difficulty to deliver the negatively charged nucleic acids to target cells. For Patisiran, the problems have been overcome by delivering the siRNA molecule in a lipid nanoparticle, able to cross the cell membranes. As transthyretin is mainly synthesized within the liver, where lipid nanoparticles naturally accumulate, the inhibition of transthyretin expression by siRNA has been successful. In a total of 148 Patisiran-treated patients compared to 77 placebo-treated patients, the almost 80% decrease in serum transthyretin levels significantly stabilized, and even improved neurological scores including gait over a 18 months period. Treatments were administered intravenously once every 3 weeks (Adams et al., 2018). Importantly, a longer-term safety and efficacy follow-up shows that patisiran maintains efficacy with an acceptable safety profile in patients with hereditary transthyretin-mediated amyloidosis (Adams et al., 2021).

Adams D et al. (2018) Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N Engl J Med 379:11-21 (

Adams D et al.  (2021) Long-term safety and efficacy of patisiran for hereditary transthyretin-mediated amyloidosis with polyneuropathy: 12-month results of an open-label extension study. Lancet Neurol 20:49-59  (

Mechanistic models of amyloid fibril formation and disease progression. a | Stability of transthyretin (TTR) homotetramers is reduced by TTR mutations, resulting in its dissociation into monomers and deposition in the extracellular spaces of systemic organs. The dissociation results in misfolding of TTR monomers and subsequent aggregation. These oligomers can be present before the completion of mature amyloid fibrils and can have toxic effects on neighbouring tissue. b | Disruption of the blood–nerve barrier (arrowhead; vessel lumen indicated by asterisk) can occur in the early phase of neuropathy , resulting in leakage of circulating TTR into the endoneurial space. Consequently, amorphous, electron- dense extracellular material that contains TTR monomers and oligomers becomes abundant in the extracellular spaces of the endoneurium. Aggregation of TTR is subsequently seen as dotty structures among amorphous materials. Finally , elongated fibrillar structures with a thickness similar to the diameter of the dotty structures are formed, leading to adjacent Schwann cell atrophy. (from Adams et al, Nat Rev Neurol, 2019)