Overwhelming data suggest that oncogenic and neurodegenerative pathways share several altered cellular responses to insults such as oxidative stress, extracellular matrix remodeling, inflammation, or cell dyscommunication. Protocadherin-10 (Pcdh10) is an adhesion molecule found to protect against tumorigenesis and essential for axonal elongation and actin dynamics during development. Here, by using genome microarrays we identified for the first time Pcdh10 up-regulation in tissues from transgenic mouse models, cultured Schwann cells, and human samples from a familial form of peripheral neuropathy (familial amyloidotic polyneuropathy). Familial amyloidotic polyneuropathy is characterized by poor functional recovery and impaired nerve regenerative response after misfolding and deposition in the peripheral nervous system of mutant transthyretin. Not only increased transcriptional and translational Pcdh10 levels occurred in axons and Schwann cells of nerves with deposited transthyretin aggregates but the pattern also extended to associated cues of axon guidance like neuropilin-1 and F-actin. These findings suggest that Pcdh10 may influence subcellular actin cytoskeletal organization and axon-axon interactions in the course of familial amyloidotic polyneuropathy. Moreover, when preventing nonfibrillar transthyretin deposition with anakinra or transthyretin siRNA, Pcdh10 protein levels were reduced, highlighting its potential as a novel disease biomarker. Whether Pcdh10 overexpression in familial amyloidotic polyneuropathy represents a protective or deleterious response, enhancing survival or promoting cell death will need further investigation.

Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1-/-) murine primary hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN's neurotrophic effects.

Although pregnancy-associated plasma protein-A (PAPP-A), a modulator of insulin-like growth factor (IGF) activity through its cleavage of IGF-binding protein (IGFBP)-4 and -5, has been known for more than two decades, knowledge about its domain architecture is still incomplete. Using position-specific iterative BLAST, we have identified distant relatives of the PAPP-A N-terminal sequence stretch of 250 residues. We present evidence that a protein domain with weak similarity to known laminin G-like (LG) modules is contained within this region, and we propose that PAPP-A and PAPP-A2 are new and unique members in the group of LG proteins as the pappalysins represent the first examples where LG modules are associated with proteinases. Fourteen beta-strands characteristic for the LG structure were tentatively located within the PAPP-A LG (PA-LG) module using secondary structure prediction and sequence alignment. Upon mammalian expression of PAPP-A truncation mutants, we defined domain boundaries showing that PA-LG is an autonomously folding unit, which spans the first 243 residues. We were unable to express PAPP-A variants which lack the PA-LG module, suggesting a possible role in stabilization of the proteolytic domain. To obtain larger amounts of protein for functional and structural analysis, the defined PA-LG domain was expressed in bacteria and folded in vitro. In addition, the availability of recombinant PA-LG module may potentially improve diagnostic assays based on the measurement of PAPP-A antigen, and also facilitate the study of PAPP-A in animal model systems.

The Na+/K+-ATPases are transmembrane ion pumps important for maintenance of ion gradients across the plasma membrane that serve to support multiple cellular functions, such as membrane potentials, regulation of cellular volume and pH, and co-transport of signaling transmitters in all animal cells. The α2Na+/K+-ATPase subunit isoform is predominantly expressed in astrocytes, which us the sharp Na+-gradient maintained by the sodium pump necessary for astroglial metabolism. Prolonged ischemia induces an elevation of [Na+]i, decreased ATP levels and intracellular pH owing to anaerobic metabolism and lactate accumulation. During ischemia, Na+/K+-ATPase-related functions will naturally increase the energy demand of the Na+/K+-ATPase ion pump. However, the role of the α2Na+/K+-ATPase in contusion injury to the spinal cord remains unknown. We used mice heterozygous mice for the loss-of-function disease-mutation G301R in the Atp1a2 gene (α 2+/G301R ) to study the effect of reduced α2Na+/K+-ATPase expression in a moderate contusion spinal cord injury (SCI) model.

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus-mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer, including acute myeloid leukaemia. However, after a high-dose AraC chemotherapy regime, patients develop severe neurotoxicity and cell death in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells. However, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesise that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukaemia patients undergoing AraC treatment. To determine the role of AraC-p75NTR signalling in the cell death of mature neurons, we used mature cerebellar granule neurons' primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurite degeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the interaction between AraC and p75NTR, we performed cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging. We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, Proline 252 and Cysteine 256 residues facilitate AraC interaction with the transmembrane domain of p75NTR resulting in uncoupling of p75NTR from the NFκB survival pathway. This, in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR. Our findings identify p75NTR as a novel molecular target to develop treatments for counteract AraC-mediated cell death of mature neurons.

The Vps10p domain receptor SorCS2 is crucial for the development and function of the nervous system and essential for brain-derived neurotrophic factor (BDNF)-induced changes in neuronal morphology and plasticity. SorCS2 regulates the subcellular trafficking of the BDNF signaling receptor TrkB as well as selected neurotransmitter receptors in a manner that is dependent on the SorCS2 intracellular domain (ICD). However, the cellular machinery and adaptor protein (AP) interactions that regulate receptor trafficking via the SorCS2 ICD are unknown. We here identify four splice variants of human SorCS2 differing in the insertion of an acidic cluster motif and/or a serine residue within the ICD. We show that each variant undergoes posttranslational proteolytic processing into a one- or two-chain receptor, giving rise to eight protein isoforms, the expression of which differs between neuronal and nonneuronal tissues and is affected by cellular stressors. We found that the only variants without the serine were able to rescue BDNF-induced branching of SorCS2 knockout hippocampal neurons, while variants without the acidic cluster showed increased interactions with clathrin-associated APs AP-1, AP-2, and AP-3. Using yeast two-hybrid screens, we further discovered that all variants bound dynein light chain Tctex-type 3; however, only variants with an acidic cluster motif bound kinesin light chain 1. Accordingly, splice variants showed markedly different trafficking properties and localized to different subcellular compartments. Taken together, our findings demonstrate the existence of eight functional SorCS2 isoforms with differential capacity for interactions with cytosolic ligands dynein light chain Tctex-type 3 and kinesin light chain 1, which potentially allows cell-type specific SorCS2 trafficking and BDNF signaling.

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin β1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.

Mutations in the neuron-specific α3 isoform of the Na+/K+-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (α3+/D801Y), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron in vivo recordings in awake α3+/D801Y mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na+/K+ exchange, but allowed the pumps to bind Na+ and become phosphorylated. These findings implicate aberrant cerebellar activity in α3 isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the α3 isoform Na+/K+-ATPase.

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels.

Nervous system development is a precisely orchestrated series of events requiring a multitude of intrinsic and extrinsic cues. Sortilin and SorCS2 are members of the Vps10p receptor family with complementary influence on some of these cues including the neurotrophins (NTs). However, the developmental time points where sortilin and SorCS2 exert their activities in conjunction or independently still remain unclear. In this study we present the characterization of the spatiotemporal expression pattern of sortilin and SorCS2 in the developing murine nervous system. Sortilin is highly expressed in the fetal nervous system with expression localized to distinct cell populations. Expression was high in neurons of the cortical plate and developing allocortex, as well as subpallial structures. Furthermore, the neuroepithelium lining the ventricles and the choroid plexus showed high expression of sortilin, together with the developing retina, spinal ganglia, and sympathetic ganglia. In contrast, SorCS2 was confined in a marked degree to the thalamus and, at E13.5, the floor plate from midbrain rostrally to spinal cord caudally. SorCS2 was also found in the ventricular zones of the ventral hippocampus and nucleus accumbens areas, in the meninges and in Schwann cells. Hence, sortilin and SorCS2 are extensively present in several distinct anatomical areas in the developing nervous system and are rarely co-expressed. Possible functions of sortilin and SorCS2 pertain to NT signaling, axon guidance and beyond. The present data will form the basis for hypotheses and study designs for unravelling the functions of sortilin and SorCS2 during the establishment of neuronal structures and connections.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1beta is primarily neurotoxic in ischemic stroke, TNF-alpha may have neurotoxic and/or neuroprotective effects. We investigated whether IL-1beta and TNF-alpha are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice.

Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies.

The Na(+)/K(+)-ATPases maintain Na(+) and K(+) electrochemical gradients across the plasma membrane, a prerequisite for electrical excitability and secondary transport in neurons. Autosomal dominant mutations in the human ATP1A3 gene encoding the neuron-specific Na(+)/K(+)-ATPase α3 isoform cause different neurological diseases, including rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC) with overlapping symptoms, including hemiplegia, dystonia, ataxia, hyperactivity, epileptic seizures, and cognitive deficits. Position D801 in the α3 isoform is a mutational hotspot, with the D801N, D801E and D801V mutations causing AHC and the D801Y mutation causing RDP or mild AHC. Despite intensive research, mechanisms underlying these disorders remain largely unknown. To study the genotype-to-phenotype relationship, a heterozygous knock-in mouse harboring the D801Y mutation (α3(+/D801Y)) was generated. The α3(+/D801Y) mice displayed hyperactivity, increased sensitivity to chemically induced epileptic seizures and cognitive deficits. Interestingly, no change in the excitability of CA1 pyramidal neurons in the α3(+/D801Y) mice was observed. The cognitive deficits were rescued by administration of the benzodiazepine, clonazepam, a GABA positive allosteric modulator. Our findings reveal the functional significance of the Na(+)/K(+)-ATPase α3 isoform in the control of spatial learning and memory and suggest a link to GABA transmission.

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as 'the master regulator of cellular anti-oxidant response', both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

Background Mutations in ATP1A2 gene encoding the Na,K-ATPase α2 isoform are associated with familial hemiplegic migraine type 2. Migraine with aura is a known risk factor for heart disease. The Na,K-ATPase is important for cardiac function, but its role for heart disease remains unknown. We hypothesized that ATP1A2 is a susceptibility gene for heart disease and aimed to assess the underlying disease mechanism. Methods and Results Mice heterozygous for the familial hemiplegic migraine type 2-associated G301R mutation in the Atp1a2 gene (α2+/G301R mice) and matching wild-type controls were compared. Reduced expression of the Na,K-ATPase α2 isoform and increased expression of the α1 isoform were observed in hearts from α2+/G301R mice (Western blot). Left ventricular dilation and reduced ejection fraction were shown in hearts from 8-month-old α2+/G301R mice (cardiac magnetic resonance imaging), and this was associated with reduced nocturnal blood pressure (radiotelemetry). Cardiac function and blood pressure of 3-month-old α2+/G301R mice were similar to wild-type mice. Amplified Na,K-ATPase-dependent Src kinase/Ras/Erk1/2 (p44/42 mitogen-activated protein kinase) signaling was observed in hearts from 8-month-old α2+/G301R mice, and this was associated with mitochondrial uncoupling (respirometry), increased oxidative stress (malondialdehyde measurements), and a heart failure-associated metabolic shift (hyperpolarized magnetic resonance). Mitochondrial membrane potential (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazolocarbocyanine iodide dye assay) and mitochondrial ultrastructure (transmission electron microscopy) were similar between the groups. Proteomics of heart tissue further suggested amplified Src/Ras/Erk1/2 signaling and increased oxidative stress and provided the molecular basis for systolic dysfunction in 8-month-old α2+/G301R mice. Conclusions Our findings suggest that ATP1A2 mutation leads to disturbed cardiac metabolism and reduced cardiac function mediated via Na,K-ATPase-dependent reactive oxygen species signaling through the Src/Ras/Erk1/2 pathway.

This article describes an immunocytochemistry (ICC) method for staining against phosphorylated forms of the kinases Akt (pAkt) and MAPK (pMAPK). Phosphorylation is induced upon their activation by a number stimuli including insulin and brain-derived neurotrophic factor (BDNF), and is prerequisite for a number of cellular processes including cell proliferation and survival [1], [2], [3], [4], [5], [6]. ICC using antibodies raised against specific phosphorylation sites allows cell-type specific and subcellular monitoring of kinase activation. Here, we test how four different antibodies against pAkt and pMAPK, respectively perform in different cell types following insulin or BDNF stimulation using different protocol conditions. We find that phospho-specific-antibodies generally perform better when using Triton X-100 as a permeabilization agent compared to Saponin. In addition, two antibodies against pAkt and two against pMAPK gave a clear increase in signal in cells stimulated with insulin or BDNF compared to the signal obtained in unstimulated cells. These antibodies also performed well when tested with western blotting. Our results illustrate that both the choice of antibody as well as protocol details are critical parameters for successful detection of phosphorylated forms of kinases by ICC. This article includes: •A protocol for subcellular detection of phosphorylated Akt and MAPK.•Validation of 8 antibodies by immunocytochemistry.•Confirmation by western blotting.

Transthyretin amyloidoses encompass a variety of acquired and hereditary diseases triggered by systemic extracellular accumulation of toxic transthyretin aggregates and fibrils, particularly in the peripheral nervous system. Since transthyretin amyloidoses are typically complex progressive disorders, therapeutic approaches aiming multiple molecular targets simultaneously, might improve therapy efficacy and treatment outcome. In this study, we evaluate the protective effect of physiologically achievable doses of curcumin on the cytotoxicity induced by transthyretin oligomers in vitro by showing reduction of caspase-3 activity and the levels of endoplasmic reticulum-resident chaperone binding immunoglobulin protein. When given to an aged Familial Amyloidotic Polyneuropathy mouse model, curcumin not only reduced transthyretin aggregates deposition and toxicity in both gastrointestinal tract and dorsal root ganglia but also remodeled congophilic amyloid material in tissues. In addition, curcumin enhanced internalization, intracellular transport and degradation of transthyretin oligomers by primary macrophages from aged Familial Amyloidotic Polyneuropathy transgenic mice, suggesting an impaired activation of naïve phagocytic cells exposed to transthyretin toxic intermediate species. Overall, our results clearly support curcumin or optimized derivatives as promising multi-target disease-modifying agent for late-stage transthyretin amyloidosis.