Somatostatin receptors and the associated intracellular machinery: the two sides of the coin.

Somatostatin receptors (SSTs) are widely expressed in pituitary tumors and neuroendocrine neoplasms (NENs) of different origins, i.e. the gastrointestinal tract and the thorax (lungs and thymus), thus representing a well-established target for medical treatment with SST ligands (SRLs). However, the response to SRLs is highly heterogeneous between tumors. Two main factors can contribute to this variability: (i) the differential SST expression among tumor types and (ii) the differential expression/modulation of the SST-related intracellular machinery. In this literature review, we provide an overview of available data on the variable expression of SSTs in pituitary tumors and NENs, together with the resulting clinical implications. Moreover, we aim to describe the complex intracellular machinery involved in SST signaling and trafficking. Particularly, we will focus on ß-arrestins and describe their role in receptor internalization and recycling, as well as the various functions of these scaffold molecules in tumor pathogenesis and progression. This review highlights the interplay between membrane receptors and intracellular machinery, together with its role in determining the clinical behavior of the tumor and the response to treatment in patients with pituitary tumors or NENs.

Levels of hormones regulating appetite and energy homeostasis in response to a 1.5-Year combined lifestyle intervention for obesity.

Background: Weight loss can induce changes in appetite-regulating hormone levels, possibly linked to increases in appetite and weight regain. However, hormonal changes vary across interventions. Here, we studied levels of appetite-regulating hormones during a combined lifestyle intervention (CLI: healthy diet, exercise and cognitive behavioral therapy). Methods: We measured levels of long-term adiposity-related hormones (leptin, insulin, high-molecular-weight (HMW) adiponectin) and short-term appetite hormones (PYY, cholecystokinin, gastric-inhibitory polypeptide, pancreatic polypeptide, FGF21, AgRP) in overnight-fasted serum of 39 patients with obesity. Hormone levels were compared between T0 (baseline), T1 (after 10 weeks) and T2 (end of treatment, 1.5 years). T0-T1 hormone changes were correlated with T1-T2 anthropometric changes. Results: Initial weight loss at T1 was maintained at T2 (-5.0%, p < 0.001), and accompanied by decreased leptin and insulin levels at T1 and T2 (all p < 0.05) compared to T0. Most short-term signals were not affected. Only PP levels were decreased at T2 compared to T0 (p < 0.05). Most changes in hormone levels during initial weight loss did not predict subsequent changes in anthropometrics, except for T0-T1 decreases in FGF21 levels and T0-T1 increases in HMW adiponectin levels tended to be associated with larger T1-T2 increases in BMI (p < 0.05 and p = 0.05, respectively). Conclusion: CLI-induced weight loss was associated with changes in levels of long-term adiposity-related hormones towards healthy levels, but not with orexigenic changes in most short-term appetite signals. Our data indicates that the clinical impact of alterations in appetite-regulating hormones during modest weight loss remains questionable. Future studies should investigate potential associations of weight-loss-induced changes in FGF21 and adiponectin levels with weight regain.

Proteomic analysis of small intestinal neuroendocrine tumors and mesenteric fibrosis.

Mesenteric metastases in small intestinal neuroendocrine tumors (SI-NETs) are associated with mesenteric fibrosis (MF) in a proportion of patients. MF can induce severe abdominal complications, and an effective preventive treatment is lacking. To elucidate possible novel therapeutic targets, we performed a proteomics-based analysis of MF. The tumor cell and stromal compartment of primary tumors and paired mesenteric metastases of SI-NET patients with MF (n = 6) and without MF (n = 6) was analyzed by liquid chromatography-mass spectrometry-based proteomics. Analysis of differential protein abundance was performed. Collagen alpha-1(XII) (COL12A1) and complement component C9 (C9) expression was evaluated by immunohistochemistry (IHC) in mesenteric metastases. A total of 2988 proteins were identified. Unsupervised hierarchical clustering showed close clustering of paired primary and mesenteric tumor cell samples. Comparing MF to non-MF samples, we detected differentially protein abundance solely in the mesenteric metastasis stroma group. There was no differential abundance of proteins in tumor cell samples or primary tumor stroma samples. Analysis of the differentially abundant proteins (n = 36) revealed higher abundance in MF samples of C9, various collagens and proteoglycans associated with profibrotic extracellular matrix dysregulation and signaling pathways. Proteins involved in fatty acid oxidation showed a lower abundance. COL12A1 and C9 were confirmed by IHC to have significantly higher expression in MF mesenteric metastases compared to non-MF. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development.

Digital quantification of somatostatin receptor subtype 2a immunostaining: a validation study.

Objective: The aim of this study was to develop an open-source and reproducible digital quantitative analysis (DIA) of somatostatin receptor subtype 2a (SST2) staining in formalin-fixed paraffin-embedded tissues of pancreatic neuroendocrine tumors (panNETs) and growth hormone (GH)-secreting pituitary adenomas (GHomas). Design: SST2 immunostaining of 18 panNETs and 39 GHomas was assessed using a novel DIA protocol and compared with a widely used semi-quantitative immunoreactivity score (IRS). Methods: The DIA software calculates the staining intensity/area and the percentage of positive cells (%PC). Four representative images were selected for each sample by two independent selectors (S1 and S2), with the analysis performed by two independent analyzers (A1 and A2). Agreement between observers was calculated using the concordance correlation coefficient (CCC). Results: In panNETs, the CCC ranged 0.935-0.977 for intensity/area and 0.942-0.983 for %PC. In GHomas, the CCC ranged 0.963-0.997 for intensity/area and 0.979-0.990 for %PC. In both panNETs and GHomas, the DIA staining intensity was strongly correlated with the IRS (Spearman rho: 0.916-0.969, P < 0.001), as well as the DIA %PC with the IRS %PC (Spearman rh: 0.826-0.881, P < 0.001). In GHomas, the biochemical response to somatostatin receptor ligands correlated with SST2 expression, evaluated both as DIA intensity/area (Spearman rho: -0.448 to -0.527, P = 0.007-0.004) and DIA %PC (Spearman rho: -0.558 to -0.644, P ≤ 0.001). Conclusions: The DIA has an excellent inter-observer agreement and showed a strong correlation with the widely used semi-quantitative IRS. The DIA protocol is an open-source, highly reproducible tool and provides a reliable quantitative evaluation of SST2 immunohistochemistry.

Obesity and Hyperphagia With Increased Defective ACTH: A Novel POMC Variant.

OBJECTIVE: Patients with pro-opiomelanocortin (POMC) defects generally present with early-onset obesity, hyperphagia, hypopigmentation and adrenocorticotropin (ACTH) deficiency. Rodent models suggest that adequate cleavage of ACTH to α-melanocortin-stimulating hormone (α-MSH) and desacetyl-α-melanocortin-stimulating hormone (d-α-MSH) by prohormone convertase 2 at the KKRR region is required for regulating food intake and energy balance. METHODS: We present 2 sisters with a novel POMC gene variant, leading to an ACTH defect at the prohormone convertase 2 cleavage site, and performed functional studies of this variant. RESULTS: The patients had obesity, hyperphagia and hypocortisolism, with markerly raised levels of ACTH but unaffected pigmentation. Their ACTH has reduced potency to stimulate the melanocortin (MC) 2 receptor, explaining their hypocortisolism. CONCLUSION: The hyperphagia and obesity support evidence that adequate cleavage of ACTH to α-MSH and d-α-MSH is also required in humans for feeding control.

Aberrant tryptophan metabolism in stromal cells is associated with mesenteric fibrosis in small intestinal neuroendocrine tumors.

Background: Increased levels of serotonin secretion are associated with mesenteric fibrosis (MF) in small intestinal neuroendocrine tumors (SI-NETs). However, the profibrotic potential of serotonin differs between patients, and in this study, we aimed to gain an understanding of the mechanisms underlying this variability. To this end, we analyzed the proteins involved in tryptophan metabolism in SI-NETs. Methods: Proteomes of tumor and stroma from primary SI-NETs and paired mesenteric metastases of patients with MF (n = 6) and without MF (n = 6) were identified by liquid chromatography-mass spectrometry (LC-MS). The differential expression of proteins involved in tryptophan metabolism between patients with and without MF was analyzed. Concurrently, monoamine oxidase A (MAO-A) expression was analyzed in the tumor and stromal compartment by immunohistochemistry (IHC) and reported as intensity over area (I/A). Results: Of the 42 proteins involved in tryptophan metabolism, 20 were detected by LC-MS. Lower abundance of ten proteins was found in mesenteric metastases stroma in patients with MF. No differential expression was found in primary SI-NETs. In patients with MF, IHC showed lower MAO-A expression in the stroma of the primary SI-NETs (median 4.2 I/A vs 6.5 I/A in patients without MF, P = 0.003) and mesenteric metastases (median 2.1 I/A vs 2.8 I/A in patients without MF, P= 0.019). Conclusion: We found a decreased expression of tryptophan and serotonin-metabolizing enzymes in the stroma in patients with MF, most notably in the mesenteric stroma. This might account for the increased profibrotic potential of serotonin and explain the variability in the development of SI-NET-associated fibrotic complications.

Sexual Dimorphism in Small-intestinal Neuroendocrine Tumors: Lower Prevalence of Mesenteric Disease in Premenopausal Women.

CONTEXT: Small-intestinal neuroendocrine tumors (SI-NETs) have a modest but significantly higher prevalence and worse prognosis in male patients. OBJECTIVE: This work aims to increase understanding of this sexual dimorphism in SI-NETs. PATIENTS AND METHODS: Retrospectively, SI-NET patients treated in a single tertiary center were included and analyzed for disease characteristics. Estrogen receptor 1 (ESR1) and 2 (ESR2), progesterone receptor (PGR), and androgen receptor (AR) messenger RNA (mRNA) expression was assessed in primary tumors and healthy intestine. Estrogen receptor alpha (ERα) and AR protein expression were analyzed by immunohistochemistry in primary tumors and mesenteric metastases. RESULTS: Of the 559 patients, 47% were female. Mesenteric metastasis/fibrosis was more prevalent in men (71% / 46%) than women (58% / 37%; P = 0.001 and P = 0.027, respectively). In women, prevalence of mesenteric metastases increased gradually with age from 41.1% in women <50 years to 71.7% in women >70 years. Increased expression of ESR1 and AR mRNA was observed in primary tumors compared to healthy intestine (both P < 0.001). ERα staining was observed in tumor cells and stroma with a strong correlation between tumor cells of primary tumors and mesenteric metastases (rho = 0.831, P = 0.02), but not in stroma (rho = -0.037, P = 0.91). AR expression was only found in stroma. CONCLUSION: Sexual dimorphism in SI-NETs was most pronounced in mesenteric disease, and the risk of mesenteric metastasis in women increased around menopause. The combination of increased ERα and AR expression in the SI-NET microenvironment suggests a modulating role of sex steroids in the development of the characteristic SI-NET mesenteric metastasis and associated fibrosis.

Reduced expression of the glucocorticoid receptor in the hippocampus of patients with drug-resistant temporal lobe epilepsy and comorbid depression.

OBJECTIVE: Depressive disorders are common among about 50% of the patients with drug-resistant temporal lobe epilepsy (TLE). The underlying etiology remains elusive, but hypothalamus-pituitary-adrenal (HPA) axis activation due to changes in glucocorticoid receptor (GR) protein expression could play an important role. Therefore, we set out to investigate expression of the GR in the hippocampus, an important brain region for HPA axis feedback, of patients with drug-resistant TLE, with and without comorbid depression. METHODS: GR expression was studied using immunohistochemistry on hippocampal sections from well-characterized TLE patients with depression (TLE + D, n = 14) and without depression (TLE - D, n = 12) who underwent surgery for drug-resistant epilepsy, as well as on hippocampal sections from autopsy control cases (n = 9). Video-electroencephalography (EEG), magnetic resonance imaging (MRI), and psychiatric and memory assessments were performed prior to surgery. RESULTS: Abundant GR immunoreactivity was present in dentate gyrus granule cells and CA1 pyramidal cells of controls. In contrast, neuronal GR expression was lower in patients with TLE, particularly in the TLE + D group. Quantitative analysis showed a smaller GR+ area in TLE + D as compared to TLE - D patients and controls. Furthermore, the ratio between the number of GR+/NeuN+ cells was lower in patients with TLE + D as compared to TLE - D and correlated negatively with the depression severity based on psychiatric history. The expression of the GR was also lower in glial cells of TLE + D compared to TLE - D patients and correlated negatively to the severity of depression. SIGNIFICANCE: Reduced hippocampal GR expression may be involved in the etiology of depression in patients with TLE and could constitute a biological marker of depression in these patients.

Expression and Cellular Distribution of P-Glycoprotein and Breast Cancer Resistance Protein in Amyotrophic Lateral Sclerosis Patients.

For amyotrophic lateral sclerosis (ALS), achieving and maintaining effective drug levels in the brain is challenging due to the activity of ATP-binding cassette (ABC) transporters which efflux drugs that affect drug exposure and response in the brain. We investigated the expression and cellular distribution of the ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) using immunohistochemistry in spinal cord (SC), motor cortex, and cerebellum from a large cohort of genetically well characterized ALS patients (n = 25) and controls (n = 14). The ALS group included 17 sporadic (sALS) and 8 familial (fALS) patients. Strong P-gp expression was observed in endothelial cells in both control and ALS specimens. Immunohistochemical analysis showed higher P-gp expression in reactive astroglial cells in both gray (ventral horn) and white matter of the SC, as well as in the motor cortex of all ALS patients, as compared with controls. BCRP expression was higher in glia in the SC and in blood vessels and glia in the motor cortex of ALS patients, as compared with controls. P-gp and BCRP immunoreactivity did not differ between sALS and fALS cases. The upregulation of both ABC transporters in the brain may explain multidrug resistance in ALS patients and has implications for the use of both approved and experimental therapeutics.

Oxidative stress and inflammation in a spectrum of epileptogenic cortical malformations: molecular insights into their interdependence.

Oxidative stress (OS) occurs in brains of patients with epilepsy and coincides with brain inflammation, and both phenomena contribute to seizure generation in animal models. We investigated whether expression of OS and brain inflammation markers co-occurred also in resected brain tissue of patients with epileptogenic cortical malformations: hemimegalencephaly (HME), focal cortical dysplasia (FCD) and cortical tubers in tuberous sclerosis complex (TSC). Moreover, we studied molecular mechanisms linking OS and inflammation in an in vitro model of neuronal function. Untangling interdependency and underlying molecular mechanisms might pose new therapeutic strategies for treating patients with drug-resistant epilepsy of different etiologies. Immunohistochemistry was performed for specific OS markers xCT and iNOS and brain inflammation markers TLR4, COX-2 and NF-κB in cortical tissue derived from patients with HME, FCD IIa, IIb and TSC. Additionally, we studied gene expression of these markers using the human neuronal cell line SH-SY5Y in which OS was induced using H2 O2 . OS markers were higher in dysmorphic neurons and balloon/giant cells in cortex of patients with FCD IIb or TSC. Expression of OS markers was positively correlated to expression of brain inflammation markers. In vitro, 100 µM, but not 50 µM, of H2 O2 increased expression of TLR4, IL-1ß and COX-2. We found that NF-κB signaling was activated only upon stimulation with 100 µM H2 O2 leading to upregulation of TLR4 signaling and IL-1ß. The NF-κB inhibitor TPCA-1 completely reversed this effect. Our results show that OS positively correlates with neuroinflammation and is particularly evident in brain tissue of patients with FCD IIb and TSC. In vitro, NF-κB is involved in the switch to an inflammatory state after OS. We propose that the extent of OS can predict the neuroinflammatory state of the brain. Additionally, antioxidant treatments may prevent the switch to inflammation in neurons thus targeting multiple epileptogenic processes at once.

MicroRNA519d and microRNA4758 can identify gangliogliomas from dysembryoplastic neuroepithelial tumours and astrocytomas.

Glioneuronal tumours, including gangliogliomas and dysembryoplastic neuroepithelial tumours, represent the most common low-grade epilepsy-associated brain tumours and are a well-recognized cause of intractable focal epilepsy in children and young adults. Classification is predominantly based on histological features, which is difficult due to the broad histological spectrum of these tumours. The aim of the present study was to find molecular markers that can be used to identify entities within the histopathology spectrum of glioneuronal tumours. The focus of this study was on microRNAs (miRNAs). miRNAs are important post-transcriptional regulators of gene expression and are involved in the pathogenesis of different neurological diseases and oncogenesis. Using a miRNA array, miR-519d and miR-4758 were found to be upregulated in gangliogliomas (n=26) compared to control cortex (n=17), peritumoural tissue (n=7), dysembryoplastic neuroepithelial tumours (n=9) and astrocytomas (grade I-IV; subependymal giant cell astrocytomas, n=10; pilocytic astrocytoma, n=15; diffuse astrocytoma grade II, n=10; grade III, n=14 and glioblastoma n=15). Furthermore, the PI3K/AKT3/P21 pathway, which is predicated to be targeted by miR-519d and miR-4758, was deregulated in gangliogliomas. Functionally, overexpression of miR-519d in an astrocytic cell line resulted in a downregulation of CDKN1A (P21) and an increase in cell proliferation, whereas co-transfection with miR-4758 counteracted this effect. These results suggest that miR-519d and miR-4758 might work in concert as regulators of the cell cycle in low grade gliomas. Furthermore, these miRNAs could be used to distinguish gangliogliomas from dysembryoplastic neuroepithelial tumours and other low and high grade gliomas and may lead to more targeted therapy.

miR147b: A novel key regulator of interleukin 1 beta-mediated inflammation in human astrocytes.

Astrocytes are important mediators of inflammatory processes in the brain and seem to play an important role in several neurological disorders, including epilepsy. Recent studies show that astrocytes produce several microRNAs, which may function as crucial regulators of inflammatory pathways and could be used as therapeutic target. We aim to study which miRNAs are produced by astrocytes during IL-1ß mediated inflammatory conditions in vitro, as well as their functional role and to validate these findings in human epileptogenic brain tissue. Sequencing was used to assess miRNA and mRNA expression in IL-1ß-stimulated human fetal astrocyte cultures. miRNAs were overexpressed in cell cultures using miRNA mimics. Expression of miRNAs in resected brain tissue from patients with tuberous sclerosis complex or temporal lobe epilepsy with hippocampal sclerosis was examined using in situ hybridization. Two differentially expressed miRNAs were found: miR146a and miR147b, which were associated with increased expression of genes related to the immune/inflammatory response. As previously reported for miR146a, overexpression of miR147b reduced the expression of the pro-inflammatory mediators IL-6 and COX-2 after IL-1ß stimulation in both astrocyte and tuberous sclerosis complex cell cultures. miR146a and miR147b overexpression decreased proliferation of astrocytes and promoted neuronal differentiation of human neural stem cells. Similarly to previous evidence for miR146a, miR147b was increased expressed in astrocytes in epileptogenic brain. Due to their anti-inflammatory effects, ability to restore aberrant astrocytic proliferation and promote neuronal differentiation, miR146a and miR147b deserve further investigation as potential therapeutic targets in neurological disorders associated with inflammation, such as epilepsy.

Amyotrophic Lateral Sclerosis, a Multisystem Pathology: Insights into the Role of TNFα.

Amyotrophic lateral sclerosis (ALS) is considered a multifactorial, multisystem disease in which inflammation and the immune system play important roles in development and progression. The pleiotropic cytokine TNFα is one of the major players governing the inflammation in the central nervous system and peripheral districts such as the neuromuscular and immune system. Changes in TNFα levels are reported in blood, cerebrospinal fluid, and nerve tissues of ALS patients and animal models. However, whether they play a detrimental or protective role on the disease progression is still not clear. Our group and others have recently reported opposite involvements of TNFR1 and TNFR2 in motor neuron death. TNFR2 mediates TNFα toxic effects on these neurons presumably through the activation of MAP kinase-related pathways. On the other hand, TNFR2 regulates the function and proliferation of regulatory T cells (Treg) whose expression is inversely correlated with the disease progression rate in ALS patients. In addition, TNFα is considered a procachectic factor with a direct catabolic effect on skeletal muscles, causing wasting. We review and discuss the role of TNFα in ALS in the light of its multisystem nature.

mTOR-related neuropathology in mutant tsc2 zebrafish: Phenotypic, transcriptomic and pharmacological analysis.

Tuberous sclerosis complex (TSC) is a rare, genetic disease caused by loss-of-function mutations in either TSC1 or TSC2. Patients with TSC are neurologically characterized by the presence of abnormal brain structure, intractable epilepsy and TSC-associated neuropsychiatric disorders. Given the lack of effective long-term treatments for TSC, there is a need to gain greater insight into TSC-related pathophysiology and to identify and develop new treatments. In this work we show that homozygous tsc2-/- mutant zebrafish larvae, but not tsc2+/- and WT larvae, display enlarged brains, reduced locomotor behavior and epileptiform discharges at 7dpf. In addition, we pharmacologically validated the TSC model by demonstrating the dramatic rescue effect of pericardially injected rapamycin, a well-known mTOR inhibitor, on selected behavioral read-outs and at the molecular level. By means of trancriptome profiling we also acquired more insight into the neuropathology of TSC, and as a result were able to highlight possible new treatment targets. The gene expression profiles of WT and tsc2+/- larvae revealed 117 differentially expressed genes (DEGs), while between WT and tsc2-/- larvae and tsc2+/- and tsc2-/- larvae there were 1414 and 1079 DEGs, respectively. Pathway enrichment analysis from the WT and tsc2-/- DEGs, identified 14 enriched pathways from the up-regulated genes and 6 enriched pathways from the down-regulated genes. Moreover, genes related to inflammation and immune response were up-regulated in the heads of tsc2-/- larvae, in line with the findings in human brain tissue where inflammatory and immune responses appear to be major hallmarks of TSC. Taken together, our phenotypic, transcriptomic and pharmacological analysis identified the tsc2-/- zebrafish as a preclinical model that mirrors well aspects of the human condition and delineated relevant TSC-related biological pathways. The model may be of value for future TSC-related drug discovery and development programs.

Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: implications for pathophysiology and treatment.

Tuberous Sclerosis Complex (TSC) is a rare genetic disorder that results from a mutation in the TSC1 or TSC2 genes leading to constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1). TSC is associated with autism, intellectual disability and severe epilepsy. Cortical tubers are believed to represent the neuropathological substrates of these disabling manifestations in TSC. In the presented study we used high-throughput RNA sequencing in combination with systems-based computational approaches to investigate the complexity of the TSC molecular network. Overall we detected 438 differentially expressed genes and 991 differentially expressed small non-coding RNAs in cortical tubers compared to autopsy control brain tissue. We observed increased expression of genes associated with inflammatory, innate and adaptive immune responses. In contrast, we observed a down-regulation of genes associated with neurogenesis and glutamate receptor signaling. MicroRNAs represented the largest class of over-expressed small non-coding RNA species in tubers. In particular, our analysis revealed that the miR-34 family (including miR-34a, miR-34b and miR-34c) was significantly over-expressed. Functional studies demonstrated the ability of miR-34b to modulate neurite outgrowth in mouse primary hippocampal neuronal cultures. This study provides new insights into the TSC transcriptomic network along with the identification of potential new treatment targets.

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.

Novel Histopathological Patterns in Cortical Tubers of Epilepsy Surgery Patients with Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is a genetic hamartoma syndrome frequently associated with severe intractable epilepsy. In some TSC patients epilepsy surgery is a promising treatment option provided that the epileptogenic zone can be precisely delineated. TSC brain lesions (cortical tubers) contain dysmorphic neurons, brightly eosinophilic giant cells and white matter alterations in various proportions. However, a histological classification system has not been established for tubers. Therefore, the aim of this study was to define distinct histological patterns within tubers based on semi-automated histological quantification and to find clinically significant correlations. In total, we studied 28 cortical tubers and seven samples of perituberal cortex from 28 TSC patients who had undergone epilepsy surgery. We assessed mammalian target of rapamycin complex 1 (mTORC1) activation, the numbers of giant cells, dysmorphic neurons, neurons, and oligodendrocytes, and calcification, gliosis, angiogenesis, inflammation, and myelin content. Three distinct histological profiles emerged based on the proportion of calcifications, dysmorphic neurons and giant cells designated types A, B, and C. In the latter two types we were able to subsequently associate them with specific features on presurgical MRI. Therefore, these histopathological patterns provide consistent criteria for improved definition of the clinico-pathological features of cortical tubers identified by MRI and provide a basis for further exploration of the functional and molecular features of cortical tubers in TSC.

Disruption of the astrocytic TNFR1-GDNF axis accelerates motor neuron degeneration and disease progression in amyotrophic lateral sclerosis.

Considerable evidence indicates that neurodegeneration in amyotrophic lateral sclerosis (ALS) can be conditioned by a deleterious interplay between motor neurons and astrocytes. Astrocytes are the major glial component in the central nervous system (CNS) and fulfill several activities that are essential to preserve CNS homeostasis. In physiological and pathological conditions, astrocytes secrete a wide range of factors by which they exert multimodal influences on their cellular neighbours. Among others, astrocytes can secrete glial cell line-derived neurotrophic factor (GDNF), one of the most potent protective agents for motor neurons. This suggests that the modulation of the endogenous mechanisms that control the production of astrocytic GDNF may have therapeutic implications in motor neuron diseases, particularly ALS. In this study, we identified TNF receptor 1 (TNFR1) signalling as a major promoter of GDNF synthesis/release from human and mouse spinal cord astrocytes in vitro and in vivo To determine whether endogenously produced TNFα can also trigger the synthesis of GDNF in the nervous system, we then focused on SOD1G93A ALS transgenic mice, whose affected tissues spontaneously exhibit high levels of TNFα and its receptor 1 at the onset and symptomatic stage of the disease. In SOD1G93A spinal cords, we verified a strict correlation in the expression of the TNFα, TNFR1 and GDNF triad at different stages of disease progression. Yet, ablation of TNFR1 completely abolished GDNF rises in both SOD1G93A astrocytes and spinal cords, a condition that accelerated motor neuron degeneration and disease progression. Our data suggest that the astrocytic TNFR1-GDNF axis represents a novel target for therapeutic intervention in ALS.

Specific pattern of maturation and differentiation in the formation of cortical tubers in tuberous sclerosis omplex (TSC): evidence from layer-specific marker expression.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multisystem disorder that results from mutations in the TSC1 or TSC2 genes, leading to constitutive activation of the mammalian target of rapamycin (mTOR) signaling pathway. Cortical tubers represent typical lesions of the central nervous system (CNS) in TSC. The pattern of cortical layering disruption observed in brain tissue of TSC patients is not yet fully understood, and little is known about the origin and phenotype of individual abnormal cell types recognized in tubers. METHODS: In the present study, we aimed to characterize dysmorphic neurons (DNs) and giant cells (GCs) of cortical tubers using neocortical layer-specific markers (NeuN, SMI32, Tbr1, Satb2, Cux2, ER81, and RORß) and to compare the features with the histo-morphologically similar focal cortical dysplasia (FCD) type IIb. We studied a cohort of nine surgically resected cortical tubers, five FCD type IIb, and four control samples using immunohistochemistry and in situ hybridization. RESULTS: Cortical tuber displayed a prominent cell loss in all cortical layers. Moreover, we observed altered proportions of layer-specific markers within the dysplastic region. DNs, in both tubers and FCD type IIb, were found positive for different cortical layer markers, regardless of their laminar location, and their immunophenotype resembles that of cortical projection neurons. CONCLUSIONS: These findings demonstrate that, similar to FCD type IIb, cortical layering is markedly disturbed in cortical tubers of TSC patients. Distribution of these disturbances is comparable in all tubers and suggests a dysmaturation affecting early and late migratory patterns, with a more severe impairment of the late stage of maturation.

Tuberous sclerosis complex neuropathology requires glutamate-cysteine ligase.

INTRODUCTION: Tuberous sclerosis complex (TSC) is a genetic disease resulting from mutation in TSC1 or TSC2 and subsequent hyperactivation of mammalian Target of Rapamycin (mTOR). Common TSC features include brain lesions, such as cortical tubers and subependymal giant cell astrocytomas (SEGAs). However, the current treatment with mTOR inhibitors has critical limitations. We aimed to identify new targets for TSC pharmacotherapy. RESULTS: The results of our shRNA screen point to glutamate-cysteine ligase catalytic subunit (GCLC), a key enzyme in glutathione synthesis, as a contributor to TSC-related phenotype. GCLC inhibition increased cellular stress and reduced mTOR hyperactivity in TSC2-depleted neurons and SEGA-derived cells. Moreover, patients' brain tubers showed elevated GCLC and stress markers expression. Finally, GCLC inhibition led to growth arrest and death of SEGA-derived cells. CONCLUSIONS: We describe GCLC as a part of redox adaptation in TSC, needed for overgrowth and survival of mutant cells, and provide a potential novel target for SEGA treatment.