Tyrosine hydroxylase and HSPB8 interact with the prion protein

The prion protein (PrP) is currently one of the most studied molecules in the neurosciences as it causes a group of neurological diseases collectively named transmissible spongiform encephalophaties (TSEs). The TSEs are characterized by a variety of motor and/or cognitive symptoms distinct from those of Parkinson and Alzheimer diseases and severely affect both humans and a variety of mammals. A great effort has thus been made to understand the molecular basis of PrP activity, both in physiological and pathological terms. In this context, the identification of neuronally-relevant interactors of PrP, capable of governing or interfering with its activity, cellular localization and/or expression, plays a crucial role. Through the expression of the proteins of interest in recombinant form in E.coli cells and the analysis of their interaction by Western blot and dot-blot, we identified two specific and neurologically relevant interactions involving the prion protein and on the one hand tyrosine hydroxylase, the enzyme that catalyzes the initial and rate-limiting reaction of the biosynthesis of catecholamines dopamine, norepinephrine and epinephrine; on the other hand the heat shock protein B8, member of the small heat shock protein family that appears to play an important role in those diseases that, like transmissible spongiform encephalopathies, are characterized by the accumulation of misfolded proteins. The association/dissociation constants of the complexes have been calculated using surface plasmon resonance and interactions were confirmed by immunohistochemistry. The data obtained show a specific and high affinity interaction (KD in the nano molar range) between the TH N-terminal regulatory domain (1-152) and the C-terminal structured domain (90-230) of PrP. The co-expression of the two proteins causes a shift in prion protein expression from a prevalent membrane-associated expression to a greater cytoplasmic localization, and also a down-regulation of the levels of expression of the prion protein without, however, affecting the topology and expression of tyrosine hydroxylase. The link between PrP and HSPB8 involves the C-terminal domain of the prion protein and the co-expression of the two proteins leads to a down-regulation of both prion protein and HSPB8. The latter, moreover, protects the prion protein from degradation by Proteinase K, while it seems to accelerate the thermal denaturation. The mutants K141E and K141N of the heat shock protein, associated with the development of neuropathy, show minor binding affinity with respect to the wild type protein and they are less effective in regulating the levels of expression, localization and degradation of the prion protein. Our results, in particular if confirmed in pathological patterns, can help to understand the physiological and pathological mechanisms of action of the prion protein and suggest tyrosine hydroxylase and heat shock protein B8 as prion protein modulators, thus as potential targets for therapeutic applications

A preclinical model for the ATLL lymphoma subtype with insights into the role of microenvironment in HTLV-1-mediated lymphomagenesis

Abstract \uef7f View references (83) Adult T cell Leukemia/Lymphoma (ATLL) is a mature T cell malignancy associated with Human T cell Leukemia Virus type 1 (HTLV-1) infection. Among its four main clinical subtypes, the prognosis of acute and lymphoma variants remains poor. The long latency (3-6 decades) and low incidence (3-5%) of ATLL imply the involvement of viral and host factors in full-blown malignancy. Despite multiple preclinical and clinical studies, the contribution of the stromal microenvironment in ATLL development is not yet completely unraveled. The aims of this study were to investigate the role of the host microenvironment, and specifically fibroblasts, in ATLL pathogenesis and to propose a murine model for the lymphoma subtype. Here we present evidence that the oncogenic capacity of HTLV-1-immortalized C91/PL cells is enhanced when they are xenotransplanted together with human foreskin fibroblasts (HFF) in immunocompromised BALB/c Rag2-/-\u3b3c -/-mice. Moreover, cell lines derived from a developed lymphoma and their subsequent in vivo passages acquired the stable property to induce aggressive T cell lymphomas. In particular, one of these cell lines, C91/III cells, consistently induced aggressive lymphomas also in NOD/SCID/IL2R\u3b3c KO (NSG) mice. To dissect the mechanisms linked to this enhanced tumorigenic ability, we quantified 45 soluble factors released by these cell lines and found that 21 of them, mainly pro-inflammatory cytokines and chemokines, were significantly increased in C91/III cells compared to the parental C91/PL cells. Moreover, many of the increased factors were also released by human fibroblasts and belonged to the known secretory pattern of ATLL cells. C91/PL cells co-cultured with HFF showed features reminiscent of those observed in C91/III cells, including a similar secretory pattern and a more aggressive behavior in vivo. On the whole, our data provide evidence that fibroblasts, one of the major stromal components, might enhance tumorigenesis of HTLV-1-infected and immortalized T cells, thus throwing light on the role of microenvironment contribution in ATLL pathogenesis. We also propose that the lymphoma induced in NSG mice by injection with C91/III cells represents a new murine preclinical ATLL model that could be adopted to test novel therapeutic interventions for the aggressive lymphoma subtype

SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8\u201310 nm) and wide (40\u201350 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER\u2013mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER\u2013mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole anim

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca2+ handling

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (A\u3b2) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca2+ handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4R\u394C20), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8-10\u202fnm) and the long- (40-50\u202fnm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca2+ homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca2+ content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4R\u394C20 tau, indicating that Ca2+ mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies

Sorcin is an early marker of neurodegeneration, Ca2+ dysregulation and endoplasmic reticulum stress associated to neurodegenerative diseases

Dysregulation of calcium signaling is emerging as a key feature in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), and targeting this process may be therapeutically beneficial. Under this perspective, it is important to study proteins that regulate calcium homeostasis in the cell. Sorcin is one of the most expressed calcium-binding proteins in the human brain; its overexpression increases endoplasmic reticulum (ER) calcium concentration and decreases ER stress in the heart and in other cellular types. Sorcin has been hypothesized to be involved in neurodegenerative diseases, since it may counteract the increased cytosolic calcium levels associated with neurodegeneration. In the present work, we show that Sorcin expression levels are strongly increased in cellular, animal, and human models of AD, PD, and HD, vs. normal cells. Sorcin partially colocalizes with RyRs in neurons and microglia cells; functional experiments with microsomes containing high amounts of RyR2 and RyR3, respectively, show that Sorcin is able to regulate these ER calcium channels. The molecular basis of the interaction of Sorcin with RyR2 and RyR3 is demonstrated by SPR. Sorcin also interacts with other ER proteins as SERCA2 and Sigma-1 receptor in a calcium-dependent fashion. We also show that Sorcin regulates ER calcium transients: Sorcin increases the velocity of ER calcium uptake (increasing SERCA activity). The data presented here demonstrate that Sorcin may represent both a novel early marker of neurodegenerative diseases and a response to cellular stress dependent on neurodegeneration

Impaired Mitochondrial ATP Production Downregulates Wnt Signaling via ER Stress Induction.

Wnt signaling affects fundamental development pathways and, if aberrantly activated, promotes the development of cancers. Wnt signaling is modulated by different factors, but whether the mitochondrial energetic state affects Wnt signaling is unknown. Here, we show that sublethal concentrations of different compounds that decrease mitochondrial ATP production specifically downregulate Wnt/β-catenin signaling in vitro in colon cancer cells and in vivo in zebrafish reporter lines. Accordingly, fibroblasts from a GRACILE syndrome patient and a generated zebrafish model lead to reduced Wnt signaling. We identify a mitochondria-Wnt signaling axis whereby a decrease in mitochondrial ATP reduces calcium uptake into the endoplasmic reticulum (ER), leading to endoplasmic reticulum stress and to impaired Wnt signaling. In turn, the recovery of the ATP level or the inhibition of endoplasmic reticulum stress restores Wnt activity. These findings reveal a mechanism that links mitochondrial energetic metabolism to the control of the Wnt pathway that may be beneficial against several pathologies

Tyrosine hydroxylase and HSPB8 interact with the prion protein

The prion protein (PrP) is currently one of the most studied molecules in the neurosciences as it causes a group of neurological diseases collectively named transmissible spongiform encephalophaties (TSEs). The TSEs are characterized by a variety of motor and/or cognitive symptoms distinct from those of Parkinson and Alzheimer diseases and severely affect both humans and a variety of mammals. A great effort has thus been made to understand the molecular basis of PrP activity, both in physiological and pathological terms. In this context, the identification of neuronally-relevant interactors of PrP, capable of governing or interfering with its activity, cellular localization and/or expression, plays a crucial role. Through the expression of the proteins of interest in recombinant form in E.coli cells and the analysis of their interaction by Western blot and dot-blot, we identified two specific and neurologically relevant interactions involving the prion protein and on the one hand tyrosine hydroxylase, the enzyme that catalyzes the initial and rate-limiting reaction of the biosynthesis of catecholamines dopamine, norepinephrine and epinephrine; on the other hand the heat shock protein B8, member of the small heat shock protein family that appears to play an important role in those diseases that, like transmissible spongiform encephalopathies, are characterized by the accumulation of misfolded proteins. The association/dissociation constants of the complexes have been calculated using surface plasmon resonance and interactions were confirmed by immunohistochemistry. The data obtained show a specific and high affinity interaction (KD in the nano molar range) between the TH N-terminal regulatory domain (1-152) and the C-terminal structured domain (90-230) of PrP. The co-expression of the two proteins causes a shift in prion protein expression from a prevalent membrane-associated expression to a greater cytoplasmic localization, and also a down-regulation of the levels of expression of the prion protein without, however, affecting the topology and expression of tyrosine hydroxylase. The link between PrP and HSPB8 involves the C-terminal domain of the prion protein and the co-expression of the two proteins leads to a down-regulation of both prion protein and HSPB8. The latter, moreover, protects the prion protein from degradation by Proteinase K, while it seems to accelerate the thermal denaturation. The mutants K141E and K141N of the heat shock protein, associated with the development of neuropathy, show minor binding affinity with respect to the wild type protein and they are less effective in regulating the levels of expression, localization and degradation of the prion protein. Our results, in particular if confirmed in pathological patterns, can help to understand the physiological and pathological mechanisms of action of the prion protein and suggest tyrosine hydroxylase and heat shock protein B8 as prion protein modulators, thus as potential targets for therapeutic applications.La proteina prionica è, ad oggi, una delle molecole più studiate nelle neuroscienze poiché essa rappresenta l’agente eziologico di un gruppo di patologie neurologiche chiamate collettivamente encefalopatie spongiformi trasmissibili (TSE). Queste patologie sono caratterizzate da una serie di sintomi motori e cognitivi distinti da quelli delle patologie di Parkinson e Alzheimer e colpiscono l’uomo e una grande varietà di mammiferi. Per tale ragione un grande sforzo è stato fatto per capire le basi molecolari dell’attività di PrP in condizioni fisiologiche e patologiche, le quali, tutt’ora, non sono del tutto comprese. In questo contesto è di cruciale importanza l’identificazione di nuovi partner di legame che regolino o interferiscano con l’attività, la localizzazione cellulare e/o l’espressione della proteina prionica. Tramite l’espressione delle proteine di interesse in forma ricombinante in cellule di E.coli e l’analisi delle loro interazioni mediante Western blot e dot-blot, abbiamo identificato due interazioni specifiche e neurologicamente rilevanti della proteina prionica coinvolgenti da un lato la tirosina idrossilasi, enzima che catalizza la reazione iniziale e limitante della biosintesi delle catecolamine dopamina, norepinefrina ed epinefrina; dall’altro l’heat shock protein B8, membro della famiglia delle small heat shock protein, che sembra ricoprire un ruolo particolarmente importante in quelle patologie che, come le encefalopatie spongiformi trasmissibili, sono caratterizzate dall’accumulo di proteine mal ripiegate. Le costanti di associazione/dissociazione dei complessi sono state calcolate mediante surface plasmon resonance e le interazioni sono state confermate tramite immunoistochimica. I dati ottenuti evidenziano un’interazione specifica e ad alta affinità (KD nel range nM) tra il dominio regolatorio N-terminale (1-152) di TH e il dominio strutturato C-terminale (90-230) di PrP. La co-espressione delle due proteine causa sia uno spostamento da un’espressione prevalentemente di membrana della proteina prionica ad una localizzazione maggiormente citoplasmatica, sia una down-regolazione dei livelli di espressione di proteina prionica senza, tuttavia, influenzare topologia ed espressione della tirosina idrossilasi. Il legame tra PrP e HSPB8 coinvolge il dominio C-terminale della proteina prionica e la co-espressione delle due proteine porta ad una down-regolazione sia di proteina prionica che di HSPB8. Quest’ultima, inoltre, protegge la proteina prionica dalla degradazione da parte di proteinasi K, mentre sembra favorirne la denaturazione termica. I mutanti K141E e K141N dell’heat shock protein, associati allo sviluppo di neuropatie, mostrano affinità di binding minori rispetto alla proteina wild type così come sono meno efficaci nel regolare i livelli di espressione, localizzazione e degradazione della proteina prionica. I nostri risultati, in particolare se confermati in modelli di patologia, possono aiutare a comprendere i meccanismi d’azione fisiologici e patologici della proteina prionica e indicano tirosina idrossilasi e heat shock protein B8 come modulatori della proteina prionica stessa e quindi come potenziali target per applicazioni terapeutiche

The Small Heat Shock Protein HspB8: Role in Nervous System Physiology and Pathology

The accumulation and aggregation of misfolded proteins can be highly cytotoxic and may underlie several human degenerative diseases characterized by neuronal inclusions such as Alzheimer's, Parkinson's, prion-like and polyglutamine repeat diseases. In this context small heat shock proteins, molecular chaperones known to be induced by cell stress, play a fundamental role by facilitating folding of nascent polypeptides, preventing aggregation of misfolded proteins and enhancing their degradation. A recently identified member of the small heat shock protein family, HspB8, is of particular interest in the field of neurological diseases since mutations in its sequence correlate with development of distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 expression has been detected in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington disease and spinocerebellar ataxia type 3. In the latter, HspB8 appears to be involved in protecting the cell from accumulation of insoluble aggregates either by preventing aggregation or by promoting degradation of improperly folded proteins. These data propose that HspB8 may be a major player in the neuroprotective response and a promising target for the development of therapeutic strategies - See more at: http://www.eurekaselect.com/123305/article#sthash.fb9jmnOq.dpu

The Close Encounter Between Alpha-Synuclein and Mitochondria

The presynaptic protein alpha-synuclein (\u3b1-syn) is unequivocally linked to the development of Parkinson's disease (PD). Not only it is the major component of amyloid fibrils found in Lewy bodies but mutations and duplication/triplication in its gene are responsible for the onset of familial autosomal dominant forms of PD. Nevertheless, the precise mechanisms leading to neuronal degeneration are not fully understood. Several lines of evidence suggest that impaired autophagy clearance and mitochondrial dysfunctions such as bioenergetics and calcium handling defects and alteration in mitochondrial morphology might play a pivotal role in the etiology and progression of PD, and indicate the intriguing possibility that \u3b1-syn could be involved in the control of mitochondrial function both in physiological and pathological conditions. In favor of this, it has been shown that a fraction of cellular \u3b1-syn can selectively localize to mitochondrial sub-compartments upon specific stimuli, highlighting possible novel routes for \u3b1-syn action. A plethora of mitochondrial processes, including cytochrome c release, calcium homeostasis, control of mitochondrial membrane potential and ATP production, is directly influenced by \u3b1-syn. Eventually, \u3b1-syn localization within mitochondria may also account for its aggregation state, making the \u3b1-syn/mitochondria intimate relationship a potential key for the understanding of PD pathogenesis. Here, we will deeply survey the recent literature in the field by focusing our attention on the processes directly controlled by \u3b1-syn within mitochondrial sub-compartments and its potential partners providing possible hints for future therapeutic targets