TrkB and gene expression

The neurotrophins are a family of secreted proteins that potently regulate diverse neuronal responses. Family members include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4/5(NT4/5). Neurotrophins bind the Trks receptor family (TrkA, B, C). NGF is the preferred ligand for TrkA, BDNF and NT4/5 are preferred for TrkB, and NT3 for TrkC, although NT3 also binds with less affinity to TrkA and TrkB. During my PhD I have focused my interest in understanding how neurotrophins regulate gene expression and, in particular, how BDNF does this through its high affinity receptor TrkB during neurogenesis. In order to answer this question, I planned to adopt the following approaches. First, to analyze global changes in gene expression after TrkB/BDNF activation, using microarray technology. Second, once a set of regulated genes was identified, to characterize the regulation of these genes at the promoter level, in order to understand which common elements are important for their regulation. The high-density oligonucleotide array of Affymetrix was performed using mRNAs that were obtained from cortical neurons of wild type mouse embryos (E15.5), and of mouse embryos possessing TrkB receptors mutated at either tyrosine 515 (trkB/shc point signaling mutants), or tyrosine 816 (trkB/plc-g point signaling mutants), or at both sites. In all cases the primary neurons were either unstimulated or stimulated with BDNF. The sensitivity of the Affymetrix system allowed me to identify a set of transcription factors that showed a higher fold induction compared to the others class of genes. This group consisted of: egr1, egr2, c-fos and mGIF/Tieg1. These genes were found to be differentially regulated in the signaling point mutant mice. Although the promoter of mGif/TIEG1 is not yet characterized and also the function of this gene is not completely clear, egr1, egr2 and c-fos are well characterized, and, several data suggest that these genes share cis acting 5’ regulatory elements. To better understand which elements and transcription factors are important for BDNF-dependent gene expression I choose the c-fos promoter as a model. Using luciferase reporter gene constructs transfected in E15.5 cortical neurons isolated from wild-type and signaling point mutant mice, I discovered that the pathways activated through the shc site promoted higher activation of c-fos promoter than pathways activated through the plc-g_site, and the two sites are both required for BDNF-dependent activation of c-fos promoter. Additionally experiments using c-fos promoter constructs, mutated at single or multiple elements, revealed that the c/ebp binding site together with the E-box are fundamental for the activation of c-fos downstream BDNF/TrkB. This result suggested that C/EBPs and bHLH transcription factors might collaborate to induce the activation of the promoter downstream of BDNF. I have demonstrated, both in vivo and in vitro, that Mash1 and NeuroD are the members of bHLH family that bind C/EBP transcription factors at different domains. That interaction is BDNF independent, and the complex is constitutively present on the c-fos promoter. The BDNF regulation of gene expression is through the post-translation modification of that complex. In fact BDNF stimulation induces an increase in C/EBPb phosphorylation on Thr188 (ERK1/2-dependent). The phosphorylation by ERK1/2 could explain the transcriptional activation of the C/EBP-Mash1-NeuroD complex downstream BDNF/TrkB. These studies identify a novel neurotrophins-regulated signaling cascade that mediates the gene expression during neurogenesis

Neurotrophin/Trk receptor signaling mediates C/EBPα, -β and NeuroD recruitment to immediate-early gene promoters in neuronal cells and requires C/EBPs to induce immediate-early gene transcription

BACKGROUND: Extracellular signaling through receptors for neurotrophins mediates diverse neuronal functions, including survival, migration and differentiation in the central nervous system, but the transcriptional targets and regulators that mediate these diverse neurotrophin functions are not well understood. RESULTS: We have identified the immediate-early (IE) genes Fos, Egr1 and Egr2 as transcriptional targets of brain derived neurotrophic factor (BDNF)/TrkB signaling in primary cortical neurons, and show that the Fos serum response element area responds to BDNF/TrkB in a manner dependent on a combined C/EBP-Ebox element. The Egr1 and Egr2 promoters contain homologous regulatory elements. We found that C/EBPα/β and NeuroD formed complexes in vitro and in vivo, and were recruited to all three homologous promoter regions. C/EBPα and NeuroD co-operatively activated the Fos promoter in transfection assays. Genetic depletion of Trk receptors led to impaired recruitment of C/EBPs and NeuroD in vivo, and elimination of Cebpa and Cebpb alleles reduced BDNF induction of Fos, Egr1 and Egr2 in primary neurons. Finally, defective differentiation of cortical dendrites, as measured by MAP2 staining, was observed in both compound Cebp and Ntrk knockout mice. CONCLUSION: We here identify three IE genes as targets for BDNF/TrkB signaling, show that C/EBPα and -β are recruited along with NeuroD to target promoters, and that C/EBPs are essential mediators of Trk signaling in cortical neurons. We show also that C/EBPs and Trks are required for cortical dendrite differentiation, consistent with Trks regulating dendritic differentiation via a C/EBP-dependent mechanism. Finally, this study indicates that BDNF induction of IE genes important for neuronal function depends on transcription factors (C/EBP, NeuroD) up-regulated during neuronal development, thereby coupling the functional competence of the neuronal cells to their differentiation

Unexpected Tolerance of α-Cleavage of the Prion Protein to Sequence Variations

The cellular form of the prion protein, PrPC, undergoes extensive proteolysis at the α site (109K↓H110). Expression of non-cleavable PrPC mutants in transgenic mice correlates with neurotoxicity, suggesting that α-cleavage is important for PrPC physiology. To gain insights into the mechanisms of α-cleavage, we generated a library of PrPC mutants with mutations in the region neighbouring the α-cleavage site. The prevalence of C1, the carboxy adduct of α-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the α-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, α-cleavage was size-dependently impaired by deletions within the domain 106–119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that α-cleavage is executed by an α-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrPC

Prion protein and Aβ-related synaptic toxicity impairment

Alzheimer's disease (AD), the most common neurodegenerative disorder, goes along with extracellular amyloid-β (Aβ) deposits. The cognitive decline observed during AD progression correlates with damaged spines, dendrites and synapses in hippocampus and cortex. Numerous studies have shown that Aβ oligomers, both synthetic and derived from cultures and AD brains, potently impair synaptic structure and functions. The cellular prion protein (PrPC) was proposed to mediate this effect. We report that ablation or overexpression of PrPC had no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of AD. These findings challenge the role of PrPC as a mediator of Aβ toxicity

Early Circulating Tumor DNA Dynamics and Efficacy of Lorlatinib in Patients With Treatment-Naive, Advanced, ALK-Positive NSCLC

Introduction: Circulating tumor DNA (ctDNA) has been used as a biomarker for prognostication and response to treatment. Here, we evaluate ctDNA as a potential biomarker for response to lorlatinib, a third-generation ALK tyrosine kinase inhibitor in patients with treatment-naive, advanced, ALK-positive NSCLC in the ongoing phase 3 CROWN study (NCT03052608). Methods: Molecular responses were calculated using mean variant allele frequency (VAF), longitudinal mean change in VAF (dVAF), and ratio to baseline. Efficacy assessments (progression-free survival [PFS] and objective response rate) were paired with individual patient ctDNA and analyzed for association. Results: Compared with baseline, mean VAF at week 4 was decreased in both treatment arms. Considering all detected somatic variants, a reduction in dVAF (≤0) was associated with a longer PFS in the lorlatinib arm. The hazard ratio (HR) for a dVAF less than or equal to 0 versus more than 0 was 0.50 (95% confidence interval [CI]: 0.23–1.12) in the lorlatinib arm. A similar association was not observed for crizotinib (HR = 1.00, 95% CI: 0.49–2.03). Comparing molecular responders with nonresponders, patients treated with lorlatinib who had a molecular response had longer PFS (HR = 0.37, 95% CI: 0.16–0.85); patients treated with crizotinib who had a molecular response had similar PFS as those without a molecular response (HR = 1.48, 95% CI: 0.67–3.30). Conclusions: In patients with treatment-naive, advanced, ALK-positive NSCLC, early ctDNA dynamics predicted better outcome with lorlatinib but not with crizotinib. These results suggest that ctDNA may be used to monitor and potentially predict efficacy of lorlatinib treatment.</p

Early Circulating Tumor DNA Dynamics and Efficacy of Lorlatinib in Patients With Treatment-Naive, Advanced, ALK-Positive NSCLC

Introduction: Circulating tumor DNA (ctDNA) has been used as a biomarker for prognostication and response to treatment. Here, we evaluate ctDNA as a potential biomarker for response to lorlatinib, a third-generation ALK tyrosine kinase inhibitor in patients with treatment-naive, advanced, ALK-positive NSCLC in the ongoing phase 3 CROWN study (NCT03052608). Methods: Molecular responses were calculated using mean variant allele frequency (VAF), longitudinal mean change in VAF (dVAF), and ratio to baseline. Efficacy assessments (progression-free survival [PFS] and objective response rate) were paired with individual patient ctDNA and analyzed for association. Results: Compared with baseline, mean VAF at week 4 was decreased in both treatment arms. Considering all detected somatic variants, a reduction in dVAF (≤0) was associated with a longer PFS in the lorlatinib arm. The hazard ratio (HR) for a dVAF less than or equal to 0 versus more than 0 was 0.50 (95% confidence interval [CI]: 0.23–1.12) in the lorlatinib arm. A similar association was not observed for crizotinib (HR = 1.00, 95% CI: 0.49–2.03). Comparing molecular responders with nonresponders, patients treated with lorlatinib who had a molecular response had longer PFS (HR = 0.37, 95% CI: 0.16–0.85); patients treated with crizotinib who had a molecular response had similar PFS as those without a molecular response (HR = 1.48, 95% CI: 0.67–3.30). Conclusions: In patients with treatment-naive, advanced, ALK-positive NSCLC, early ctDNA dynamics predicted better outcome with lorlatinib but not with crizotinib. These results suggest that ctDNA may be used to monitor and potentially predict efficacy of lorlatinib treatment.</p

The Comprehensive Native Interactome of a Fully Functional Tagged Prion Protein

The enumeration of the interaction partners of the cellular prion protein, PrPC, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrPC. When expressed in transgenic mice, PrPmyc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrPC. PrPmyc antagonized the toxicity of truncated PrP, restored prion infectibility of PrPC-deficient mice, and was physically incorporated into PrPSc aggregates, indicating that it possessed all functional characteristics of genuine PrPC. We then immunopurified myc epitope-containing protein complexes from PrPmyc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrPC and may represent component of a multiprotein complex. Selected PrPC interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Canagliflozin and Cardiovascular and Renal Outcomes in Type 2 Diabetes Mellitus and Chronic Kidney Disease in Primary and Secondary Cardiovascular Prevention Groups

Background: Canagliflozin reduces the risk of kidney failure in patients with type 2 diabetes mellitus and chronic kidney disease, but effects on specific cardiovascular outcomes are uncertain, as are effects in people without previous cardiovascular disease (primary prevention). Methods: In CREDENCE (Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation), 4401 participants with type 2 diabetes mellitus and chronic kidney disease were randomly assigned to canagliflozin or placebo on a background of optimized standard of care. Results: Primary prevention participants (n=2181, 49.6%) were younger (61 versus 65 years), were more often female (37% versus 31%), and had shorter duration of diabetes mellitus (15 years versus 16 years) compared with secondary prevention participants (n=2220, 50.4%). Canagliflozin reduced the risk of major cardiovascular events overall (hazard ratio [HR], 0.80 [95% CI, 0.67-0.95]; P=0.01), with consistent reductions in both the primary (HR, 0.68 [95% CI, 0.49-0.94]) and secondary (HR, 0.85 [95% CI, 0.69-1.06]) prevention groups (P for interaction=0.25). Effects were also similar for the components of the composite including cardiovascular death (HR, 0.78 [95% CI, 0.61-1.00]), nonfatal myocardial infarction (HR, 0.81 [95% CI, 0.59-1.10]), and nonfatal stroke (HR, 0.80 [95% CI, 0.56-1.15]). The risk of the primary composite renal outcome and the composite of cardiovascular death or hospitalization for heart failure were also consistently reduced in both the primary and secondary prevention groups (P for interaction &gt;0.5 for each outcome). Conclusions: Canagliflozin significantly reduced major cardiovascular events and kidney failure in patients with type 2 diabetes mellitus and chronic kidney disease, including in participants who did not have previous cardiovascular disease