The immune system in Parkinson's disease: what we know so far.

Parkinson's disease is characterized neuropathologically by the degeneration of dopaminergic neurons in the ventral midbrain, the accumulation of α-synuclein (α-syn) aggregates in neurons and chronic neuroinflammation. In the past two decades, in vitro, ex vivo and in vivo studies have consistently shown the involvement of inflammatory responses mediated by microglia and astrocytes, which may be elicited by pathological α-syn or signals from affected neurons and other cell types, and are directly linked to neurodegeneration and disease development. Apart from the prominent immune alterations seen in the CNS, including the infiltration of T cells into the brain, more recent studies have demonstrated important changes in the peripheral immune profile within both the innate and adaptive compartments, particularly involving monocytes, CD4+ and CD8+ T cells.

MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying mechanisms remain incompletely understood. In this work, we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with an unknown physiological substrate, displays an immune function by controlling inflammatory and type-I interferon (IFN) responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as an effector protein regulated by MOK, by promoting Ser-phospho-Brd4 levels.

Immune Signaling Kinases in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).

Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disorder of motor neurons in adults, with a median survival of 3-5 years after appearance of symptoms, and with no curative treatment currently available. Frontotemporal dementia (FTD) is also an adult-onset neurodegenerative disease, displaying not only clinical overlap with ALS, but also significant similarities at genetic and pathologic levels. Apart from the progressive loss of neurons and the accumulation of protein inclusions in certain cells and tissues, both disorders are characterized by chronic inflammation mediated by activated microglia and astrocytes, with an early and critical impact of neurodegeneration along the disease course.

Distinct responses of human peripheral blood cells to different misfolded protein oligomers.

Increasing evidence indicates that peripheral immune cells play a prominent role in neurodegeneration connected to protein misfolding, which are associated with formation of aberrant aggregates, including soluble protein misfolded oligomers. The precise links, however, between the physicochemical features of diverse oligomers and their effects on the immune system, particularly on adaptive immunity, remain currently unexplored, due partly to the transient and heterogeneous nature of the oligomers themselves. To overcome these limitations, we took advantage of two stable and well-characterized types of model oligomers (A and B), formed by HypF-N bacterial protein, type B oligomers displaying lower solvent-exposed hydrophobicity.

Differential Interactome and Innate Immune Response Activation of Two Structurally Distinct Misfolded Protein Oligomers.

The formation of misfolded protein oligomers during early stages of amyloid aggregation and the activation of neuroinflammatory responses are two key events associated with neurodegenerative diseases. Although it has been established that misfolded oligomers are involved in the neuroinflammatory process, the links between their structural features and their functional effects on the immune response remain unknown. To explore such links, we took advantage of two structurally distinct soluble oligomers (type A and B) of protein HypF-N and compared the elicited microglial inflammatory responses.

Immunization with α-synuclein/Grp94 reshapes peripheral immunity and suppresses microgliosis in a chronic Parkinsonism model.

Neuroinflammation mediated by chronically activated microglia, largely caused by abnormal accumulation of misfolded α-synuclein (αSyn) protein, is known to contribute to the pathophysiology of Parkinson's disease (PD). In this work, based on the immunomodulatory activities displayed by particular heat-shock proteins (HSPs), we tested a novel vaccination strategy that used a combination of αSyn and Grp94 (HSPC4 or Gp96) chaperone and a murine PD model. We used two different procedures, first, the adoptive transfer of splenocytes from αSyn/Grp94-immunized mice to recipient animals, and second, direct immunization with αSyn/Grp94, to study the effects in a chronic mouse MPTP-model of parkinsonism.

Extracellular TDP-43 aggregates target MAPK/MAK/MRK overlapping kinase (MOK) and trigger caspase-3/IL-18 signaling in microglia.

Dysregulated microglial responses are central in neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar disease (FTLD). Pathologic TDP-43, which is typically found in intracellular inclusions, is a misfolding protein with emerging roles in ALS and FTLD. Recently, TDP-43 species have been found in extracellular fluids of patients; however, the overall implications of TDP-43-mediated signaling linked to neuroinflammation are poorly understood.

The chaperonin CCT inhibits assembly of α-synuclein amyloid fibrils by a specific, conformation-dependent interaction.

The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington's disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question.

The 'Omics' of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease that primarily affects motor neurons and is accompanied by sustained unregulated immune responses, but without clear indications of the ultimate causative mechanisms. The identification of a diverse array of ALS phenotypes, a series of recently discovered mutations, and the links between ALS and frontotemporal degeneration have significantly increased our knowledge of the disease. In this review we discuss the main features involved in ALS pathophysiology in the context of recent advances in 'omics' approaches, including genomics, proteomics, and others.

Chaperome screening leads to identification of Grp94/Gp96 and FKBP4/52 as modulators of the α-synuclein-elicited immune response.

We have investigated the potential role of molecular chaperones as modulators of the immune response by using α-synuclein (αSyn) as an aggregation-prone model protein. We first performed an in vitro immunoscreening with 21 preselected candidate chaperones and selected 2 from this set as displaying immunological activity with differential profiles, Grp94/Gp96 and FKBP4/52. We then immunized mice with both chaperone/α-synuclein combinations using monomeric or oligomeric α-synuclein (MαSyn or OαSyn, respectively), and we characterized the immune response generated in each case.

Chaperoned amyloid proteins for immune manipulation: α-Synuclein/Hsp70 shifts immunity toward a modulatory phenotype.

α-Synuclein (αSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson's disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective αSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical "chaperone" activities and their recently discovered and diverse "immunoactive" properties.

Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation.

We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures.

Preconditioning of microglia by α-synuclein strongly affects the response induced by toll-like receptor (TLR) stimulation.

In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson's disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer's disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders.

Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.

Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers.

A rationally designed six-residue swap generates comparability in the aggregation behavior of α-synuclein and β-synuclein.

The aggregation process of α-synuclein, a protein closely associated with Parkinson's disease, is highly sensitive to sequence variations. It is therefore of great importance to understand the factors that define the aggregation propensity of specific mutational variants as well as their toxic behavior in the cellular environment. In this context, we investigated the extent to which the aggregation behavior of α-synuclein can be altered to resemble that of β-synuclein, an aggregation-resistant homologue of α-synuclein not associated with disease, by swapping residues between the two proteins.

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers.

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol.

Glial innate immunity generated by non-aggregated alpha-synuclein in mouse: differences between wild-type and Parkinson's disease-linked mutants.

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma.

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/alpha-synuclein complex by Hip.

The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of alpha-synuclein (alphaSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides.

Immunological features of alpha-synuclein in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence, in the brain, of intra-cellular protein inclusions highly enriched in aggregated alpha-synuclein (alphaSyn), known as Lewy bodies. The onset of PD is accompanied by a local immune reaction in regions of the brain affected by the inclusions, although the mechanism that leads to pathogenesis is far from clear. It is, however, established that disease onset and progression are characterized by sustained activation of microglia, which is linked to significant dopaminergic neuron loss in the substantia nigra.

The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase.

In essence, evolutionary processes occur gradually, while maintaining fitness throughout. Along this line, it has been proposed that the ability of a progenitor to promiscuously catalyze a low level of the evolving activity could facilitate the divergence of a new function by providing an immediate selective advantage. To directly establish a role for promiscuity in the divergence of natural enzymes, we attempted to trace the origins of a bacterial phosphotriesterase (PTE), an enzyme thought to have evolved for the purpose of degradation of a synthetic insecticide introduced in the 20th century.

Enzyme promiscuity: evolutionary and mechanistic aspects.

The past few years have seen significant advances in research related to the 'latent skills' of enzymes - namely, their capacity to promiscuously catalyze reactions other than the ones they evolved for. These advances regard (i) the mechanism of catalytic promiscuity - how enzymes, that generally exert exquisite specificity, promiscuously catalyze other, and sometimes barely related, reactions; (ii) the evolvability of promiscuous functions - namely, how latent activities evolve further, and in particular, how promiscuous activities can firstly evolve without severely compromising the original activity. These findings have interesting implications on our understanding of how new enzymes evolve.

Use of full-length recombinant calflagin and its c fragment for improvement of diagnosis of Trypanosoma cruzi infection.

Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T.

Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily.

The amidohydrolase superfamily comprises hundreds of hydrolytic enzymes of the (beta/alpha)8 barrel fold with mono- or binuclear active-site metal centers, and a diverse spectrum of substrates and reactions. Promiscuous activities, or cross-reactivities, between different members of the same superfamily may provide important hints regarding evolutionary and mechanistic relationships. We examined three members: dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP).