Oligomeric α-Synuclein induces skin degeneration in reconstructed human epidermis.

Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1ß and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1ß inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.

WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that lead to damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins six, eight, 18 and tumor necrosis factor-alpha (TNF-α) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

SARS-CoV-2 infection of the central nervous system in a 14-month-old child: A case report of a complete autopsy.

BACKGROUND: Neurological and other systemic complications occur in adults with severe COVID-19. Here we describe SARS-CoV-2 infection complicated by neuroinvasion in the post-mortem tissues of a child. METHODS: We performed a complete autopsy of a 14-month-old child who died of COVID-19 pneumonitis. Histological sections of multiple organs were stained with haematoxylin and eosin. Luxol fast blue staining for myelin and immunohistochemistry were performed in selected areas of the brain. The presence of SARS-CoV-2 was investigated by immunostaining with anti-spike protein antibody and by RT-qPCR. FINDINGS: Lesions included microthrombosis, pulmonary congestion, interstitial oedema, lymphocytic infiltrates, bronchiolar injury, collapsed alveolar spaces, cortical atrophy, and severe neuronal loss. SARS-CoV-2 staining was observed along the apical region of the choroid plexus (ChP) epithelium and in ependymal cells of the lateral ventricle, but was restricted to ChP capillaries and vessels in some regions. SARS-CoV-2 infection of brain tissue was confirmed by RT-qPCR in fragments of the ChP, lateral ventricle, and cortex. INTERPRETATION: Our results show multisystemic histopathological alterations caused by SARS-CoV-2 infection and contribute to knowledge regarding the course of fatal COVID-19 in children. Furthermore, our findings of ChP infection and viral neurotropism suggest that SARS-CoV-2 may invade the central nervous system by blood-cerebrospinal fluid barrier disruption. FUNDING: Carlos Chagas Filho Foundation for Supporting Research in the State of Rio de Janeiro (FAPERJ); the National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES), in addition to intramural grants from D'Or Institute for Research and Education. EDITOR'S NOTE: This translation in Portuguese was submitted by the authors and we reproduce it as supplied. It has not been peer reviewed. Our editorial processes have only been applied to the original abstract in English, which should serve as reference for this manuscript. RESUMO: Complicações sistêmicas e neurológicas foram descritas em adultos com COVID-19 grave. Neste trabalho, descrevemos a infecção por SARS-CoV-2, incluindo sua neuroinvasão, nos tecidos post-mortem de uma criança. MÉTODOS: Realizamos a autópsia completa de uma criança de 14 meses que morreu de pneumonite por COVID-19. Cortes histológicos de múltiplos órgãos foram corados com Hematoxilina e Eosina. A coloração de Luxol Fast Blue para mielina e imuno-histoquímica foram realizadas em áreas selecionadas do cérebro. A presença de SARS-CoV-2 foi investigada por imunomarcação com anticorpo anti-proteína spike e por RT-qPCR. ACHADOS: As lesões incluíram microtrombose, congestão pulmonar, edema intersticial, infiltrados linfocíticos, lesão bronquiolar, colapso dos espaços alveolares, atrofia cortical e perda neuronal grave. A presença de SARS-CoV-2 foi observada ao longo da região apical do epitélio do plexo coróide (PC) e nas células ependimárias do ventrículo lateral, mas ficou restrita aos capilares e vasos do PC em outras regiões. A infecção do tecido cerebral por SARS-CoV-2 foi confirmada por RT-qPCR em fragmentos do PC, ventrículo lateral e cortex cerebral. INTERPRETAÇÃO: Nossos resultados mostram alterações histopatológicas multissistêmicas causadas pela infecção por SARS-CoV-2 e contribuem para ampliar o conhecimento sobre a evolução da COVID-19 fatal em crianças. Além disso, nossos achados sobre a infecção no PC e neurotropismo viral sugerem que o SARS-CoV-2 pode invadir o sistema nervoso central pela ruptura da barreira sangue-líquido cefalorraquidiano. FINANCIAMENTO: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) e Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), além de financiamento intramural do Instituto D'Or de Pesquisa e Educação.

Non-permissive SARS-CoV-2 infection in human neurospheres.

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres.

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.

Non-permissive SARS-CoV-2 infection in human neurospheres.

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

Grafts of human adipose-derived stem cells into a biodegradable poly (acid lactic) conduit enhances sciatic nerve regeneration.

Despite the regenerative potential of the Peripheral Nervous System (PNS), injuries with loss of a nerve segment make the functional recovery a challenge. This work aimed to investigate the effects of the association of biodegradable conduits of poly (lactic acid) (PLA) with human adipose-derived stem cells (hADSCs) on the regeneration of the sciatic nerve. C57BL / 6 male mice were submitted to sciatic nerve transection followed by tubulization with PLA conduit. Animals were allocated in two groups: the first received an injection of DMEM inside the conduit (DMEM) and the second received hADSCs inside it (hADSC). Sensory and motor functions were assessed by the pinprick test and electroneuromiography, respectively. To assess neuronal survival the retrograde tracer fluorogold was injected into the sciatic nerve distally to the lesion site. One week after that, animals were sacrificed, tissues harvested and processed for morphological evaluation. After eight weeks, all animals showed sensory recovery in the pinprick test and there was no significant difference between the two groups. The amplitude of the compound muscle action potential was higher in the hADSCs group. The number of myelinated nerve fibers, muscle cells and motor plates was higher in the hADSC group. There was also greater survival of sensory and motor neurons in the hADSC animals. These results suggest that the association of PLA conduit and cell therapy with hADSCs leads to a better functional and morphological recovery after sciatic nerve transection.

Naringenin complexed with hydroxypropyl-ß-cyclodextrin improves the sciatic nerve regeneration through inhibition of p75NTR and JNK pathway.

Peripheral nerve injuries are common conditions that often lead to dysfunctions. Although much knowledge exists on the several factors that mediate the complex biological process involved in peripheral nerve regeneration, there is a lack of effective treatments that ensure full functional recovery. Naringenin (NA) is the most abundant flavanone found in citrus fruits and it has promising neuroprotective, anti-inflammatory and antioxidant effects. This study aimed to enhance peripheral nerve regeneration using an inclusion complex containing NA and hydroxypropyl-ß-cyclodextrin (HPßCD), named NA/HPßCD. A mouse sciatic nerve crush model was used to evaluate the effects of NA/HPßCD on nerve regeneration. Sensory and motor parameters, hyperalgesic behavior and the sciatic functional index (SFI), respectively, improved with NA treatment. Western blot analysis revealed that the levels of p75NTR ICD and p75NTR full length as well phospho-JNK/total JNK ratios were preserved by NA treatment. In addition, NA treatment was able to decrease levels of caspase 3. The concentrations of TNF-α and IL-1ß were decreased in the lumbar spine, on the other hand there was an increase in IL-10. NA/HPßCD presented a better overall morphological profile but it was not able to increase the number of myelinated fibers. Thus, NA was able to enhance nerve regeneration, and NA/HPßCD decreased effective drug doses while maintaining the effect of the pure drug, demonstrating the advantage of using the complex over the pure compound.

Synthesis and evaluation of the mutagenicity of 3-alkylpyridine marine alkaloid analogues with anticancer potential.

Theonella sp is an important source of biologically-active 3-alkylpyridine alkaloids (3-APAs) that has shown a wide variety of promising biological effects. In the present work, two new 3-APAs analogues were synthesized based on molecular modeling studies to act as potential antimalarial agents. These theoneladin C analogues, containing the thiocyanate group in their chemical structures, were synthesized and evaluated against Plasmodium falciparum (IC50 values ranging from 2.3 to 5.5µM). The structural and energetic analysis demonstrated a high chemical affinity of the two analogues for their target, the heme group. However, despite the good antimalarial activity, the compounds exhibited high cytotoxicity and a lack of selectivity for human cell lines. These findings prompted us to evaluate the cytotoxicity of these compounds against human cancer cell lines. In order to better understand the mechanisms responsible for the toxicity, a variety of genotoxicity assays were performed in vitro. One of the compounds assayed presented an interesting selectivity and high toxicity to the human colon cancer cell line RKO-AS45-1. In addition, the results of the micronucleus assay, comet assay, Ames assay and annexin-V/propidium iodide staining showed that the synthetic alkaloids were able to induce chromosomal mis-segregation and trigger cell death by apoptosis. These results demonstrate that the compounds assessed herein may be promising prototypes of anticancer chemotherapeutic agents.

Long-Chain Omega-3 Fatty Acids Supplementation Accelerates Nerve Regeneration and Prevents Neuropathic Pain Behavior in Mice.

Fish oil (FO) is the main source of long chain omega-3 polyunsaturated fatty acids (ω-3 PUFAs), which display relevant analgesic and anti-inflammatory properties. Peripheral nerve injury is driven by degeneration, neuroinflammation, and neuronal plasticity which results in neuropathic pain (NP) symptoms such as allodynia and hyperalgesia. We tested the preventive effect of an EPA/DHA-concentrate fish oil (CFO) on NP development and regenerative features. Swiss mice received daily oral treatment with CFO 4.6 or 2.3 g/kg for 10 days after NP was induced by partial sciatic nerve ligation. Mechanical allodynia and thermal hypernociception were assessed 5 days after injury. CFO 2.3 g/kg significantly prevented mechanical and thermal sensitization, reduced TNF levels in the spinal cord, sciatic MPO activity, and ATF-3 expression on DRG cells. CFO improved Sciatic Functional Index (SFI) as well as electrophysiological recordings, corroborating the increased GAP43 expression and total number of myelinated fibers observed in sciatic nerve. No locomotor activity impairment was observed in CFO treated groups. These results point to the regenerative and possibly protective properties of a combined EPA and DHA oral administration after peripheral nerve injury, as well as its anti-neuroinflammatory activity, evidencing ω-3 PUFAs promising therapeutic outcomes for NP treatment.

Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts.

Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes, but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. We identified five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons. These recapitulated the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons, as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibited TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we found that the technique was able to reveal previously unknown aspects of human disease phenotypes in vitro.

Enhancement of median nerve regeneration by mesenchymal stem cells engraftment in an absorbable conduit: improvement of peripheral nerve morphology with enlargement of somatosensory cortical representation.

We studied the morphology and the cortical representation of the median nerve (MN), 10 weeks after a transection immediately followed by treatment with tubulization using a polycaprolactone (PCL) conduit with or without bone marrow-derived mesenchymal stem cell (MSC) transplant. In order to characterize the cutaneous representation of MN inputs in primary somatosensory cortex (S1), electrophysiological cortical mapping of the somatosensory representation of the forepaw and adjacent body parts was performed after acute lesion of all brachial plexus nerves, except for the MN. This was performed in ten adult male Wistar rats randomly assigned in three groups: MN Intact (n = 4), PCL-Only (n = 3), and PCL+MSC (n = 3). Ten weeks before mapping procedures in animals from PCL-Only and PCL+MSC groups, animal were subjected to MN transection with removal of a 4-mm-long segment, immediately followed by suturing a PCL conduit to the nerve stumps with (PCL+MSC group) or without (PCL-Only group) injection of MSC into the conduit. After mapping the representation of the MN in S1, animals had a segment of the regenerated nerve processed for light and transmission electron microscopy. For histomorphometric analysis of the nerve segment, sample size was increased to five animals per experimental group. The PCL+MSC group presented a higher number of myelinated fibers and a larger cortical representation of MN inputs in S1 (3,383 ± 390 fibers; 2.3 mm(2), respectively) than the PCL-Only group (2,226 ± 575 fibers; 1.6 mm(2)). In conclusion, MSC-based therapy associated with PCL conduits can improve MN regeneration. This treatment seems to rescue the nerve representation in S1, thus minimizing the stabilization of new representations of adjacent body parts in regions previously responsive to the MN.

High- and low-frequency transcutaneous electrical nerve stimulation delay sciatic nerve regeneration after crush lesion in the mouse.

The stimulation of peripheral nerve regeneration has been studied in different ways, including the use of electrical fields. The capacity of this modality to enhance nerve regeneration is influenced by the parameters used, including current type, frequency, intensity, and means of administration. Transcutaneous electrical nerve stimulation (TENS) is a frequently used form of administering electrical current to the body, but its effects on peripheral nerve regeneration are not known. This study assessed the influence of TENS on sciatic nerve regeneration, using a model of crush lesion in the mouse. Mice were stimulated 30 min a day, 5 days a week, for 5 weeks with both high- (100 Hz) and low- (4 Hz) frequency TENS. Control animals had the sciatic nerve crushed but were not stimulated. Assessment was performed weekly by functional analysis using the Static Sciatic Index for the mouse and at the end of the experiment by light and electron microscopy. The results showed that although there were no differences between the groups regarding the Static Sciatic Index values, TENS led to nerves with morphological signs of impaired regeneration. At light microscopy level, TENS nerves presented more axons with dark axoplasm, signs of edema, and a less organized cytoarchitecture. Electronmicrographs showed fewer and thinner thick myelinated fibers and increased number of Schwann cell nuclei. Myelinated axon diameters and density and diameter of nonmyelinated fibers were not affected by TENS, leading to the conclusion that this regimen of electrical stimulation leads to a delayed regeneration after a crush lesion of the sciatic nerve in the mouse. All these effects were more pronounced on high-frequency TENS nerves.