Bredford Kerr

Regulation of Memory Formation by the Transcription Factor XBP1

Contextual memory formation relies on the induction of new genes in the hippocampus. A polymorphism in the promoter of the transcription factor XBP1 was identified as a risk factor for Alzheimers disease and bipolar disorders. XBP1 is a major regulator of the unfolded protein response (UPR), mediating adaptation to endoplasmic reticulum (ER) stress. Using a phenotypic screen, we uncovered an unexpected function of XBP1 in cognition and behavior. Mice lacking XBP1 in the nervous system showed specific impairment of contextual memory formation and long-term potentiation (LTP), whereas neuronal XBP1s overexpression improved performance in memory tasks. Gene expression analysis revealed that XBP1 regulates a group of memory-related genes, highlighting brain-derived neurotrophic factor (BDNF), a key component in memory consolidation. Overexpression of BDNF in the hippocampus reversed the XBP1-deficient phenotype. Our study revealed an unanticipated function of XBP1 in cognitive processes that is apparently unrelated to its role in ER stress.

NTRK1 and NTRK2 receptors facilitate follicle assembly and early follicular development in the mouse ovary.

Recent studies have demonstrated that neurotrophins (NTs) and their NTRK tyrosine kinase receptors, thought to be exclusively required for the development of the nervous system, are also involved in controlling ovarian development. Here, we show that primordial follicle formation is decreased in the absence of nerve growth factor (NGF) or its receptor NTRK1, and in the absence of NTRK2, the receptor for neurotrophin-4 (NTF4) and brain-derived neurotrophic factor (BDNF). This deficiency is not due to premature oocyte loss, because the ovaries of Ntrk1(-/-) and Ntrk2(-/-) mice do not show an increased rate of oocyte death antedating the initiation of folliculogenesis. Moreover, exposure of NGF-deficient ovaries to NGF rescues the defect in follicular assembly, if NTRK1 receptors are present, suggesting that the absence of NTs causes a delay, and not an irretrievable loss, of follicle formation. Both the number of secondary follicles and FSH receptor (FSHR) expression are diminished in Ntrk1- and Ntrk2-null ovaries, but not in ovaries lacking the common NT receptor NGFR. Transient exposure of wild-type ovaries to NTF4 increases Fshr gene expression and enhances the ability of the ovary to respond to FSH with formation of cyclin D2, a cell cycle protein mediating the proliferative actions of FSH in the ovary. These results indicate that both NTRK1 and NTRK2 receptors are necessary for the timely assembly of primordial follicles and for sustaining early follicular development. They also suggest that a mechanism by which NTRK2 receptors facilitate subsequent follicle development is by inducing the formation of functional FSHR.

Unconventional Transcriptional Response to Environmental Enrichment in a Mouse Model of Rett Syndrome

Background Rett syndrome (RTT) is an X-linked postnatal neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2) and one of the leading causes of mental retardation in females. RTT is characterized by psychomotor retardation, purposeless hand movements, autistic-like behavior and abnormal gait. We studied the effects of environmental enrichment (EE) on the phenotypic manifestations of a RTT mouse model that lacks MeCP2 (Mecp2 −/y). Principal Findings We found that EE delayed and attenuated some neurological alterations presented by Mecp2 −/y mice and prevented the development of motor discoordination and anxiety-related abnormalities. To define the molecular correlate of this beneficial effect of EE, we analyzed the expression of several synaptic marker genes whose expression is increased by EE in several mouse models. Conclusions/Significance We found that EE induced downregulation of several synaptic markers, suggesting that the partial prevention of RTT-associated phenotypes is achieved through a non-conventional transcriptional program.

Loss of Ntrk2/Kiss1r Signaling in Oocytes Causes Premature Ovarian Failure

Neurotrophins (NTs), once believed to be neural-specific trophic factors, are now known to also provide developmental cues to non-neural cells. In the ovary, NTs contribute to both the formation and development of follicles. Here we show that oocyte-specific deletion of the Ntrk2 gene that encodes the NTRK2 receptor (NTRK2) for neurotrophin-4/5 and brain-derived neurotrophic factor (BDNF) results in post-pubertal oocyte death, loss of follicular organization, and early adulthood infertility. Oocytes lacking NTRK2 do not respond to gonadotropins with activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mediated signaling. Before puberty, oocytes only express a truncated NTRK2 form (NTRK2.T1), but at puberty full-length (NTRK2.FL) receptors are rapidly induced by the preovulatory gonadotropin surge. A cell line expressing both NTRK2.T1 and the kisspeptin receptor (KISS1R) responds to BDNF stimulation with activation of Ntrk2 expression only if kisspeptin is present. This suggests that BDNF and kisspeptin that are produced by granulosa cells (GCs) of periovulatory follicles act in concert to mediate the effect of gonadotropins on Ntrk2 expression in oocytes. In keeping with this finding, the oocytes of NTRK2-intact mice fail to respond to gonadotropins with increased Ntrk2 expression in the absence of KISS1R. Our results demonstrate that the preovulatory gonadotropin surge promotes oocyte survival at the onset of reproductive cyclicity by inducing oocyte expression of NTRK2.FL receptors that set in motion an AKT-mediated survival pathway. They also suggest that gonadotropins activate NTRK2.FL expression via a dual communication pathway involving BDNF and kisspeptin produced in GCs and their respective receptors NTRK2.T1 and KISS1R expressed in oocytes.

Transgenic complementation of MeCP2 deficiency: phenotypic rescue of Mecp2-null mice by isoform-specific transgenes

Rett syndrome (RTT) is a disorder that affects patients’ ability to communicate, move and behave. RTT patients are characterized by impaired language, stereotypic behaviors, frequent seizures, ataxia and sleep disturbances, with the onset of symptoms occurring after a period of seemingly normal development. RTT is caused by mutations in methyl-CpG binding protein 2 (MECP2), an X-chromosome gene encoding for MeCP2, a protein that regulates gene expression. MECP2 generates two alternative splice variants encoding two protein isoforms that differ only in the N-terminus. Although no functional differences have been identified for these splice variants, it has been suggested that the RTT phenotype may occur in the presence of a functional MeCP2-e2 protein. This suggests that the two isoforms might be functionally distinct. Supporting this notion, the two variants show regional and age-related differences in transcript abundance. Here, we show that transgenic expression of either the MeCP2-e1 or MeCP2-e2 splice variant results in prevention of development of RTT-like phenotypic manifestations in a mouse model lacking Mecp2. Our results indicate that the two MeCP2 splice variants can substitute for each other and fulfill the basic functions of MeCP2 in the mouse brain.

Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury.

Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.

Brain-derived neurotrophic factor regulates the expression of D1 dopamine receptors

We have previously demonstrated that the CAD catecholaminergic neuronal cell line is an appropriate model system to study the regulation of D1 dopamine receptor expression. In this report, we show that brain‐derived neurotrophic factor (BDNF) up‐regulates the expression of D1 dopamine receptor in CAD cells. In addition, by comparing D1 receptor mRNA expression in wild‐type, heterozygous and homozygous trkB knockout mice, we show that TrkB receptor signaling up‐regulates D1 receptor expression in vivo. In CAD cells expressing the TrkB receptor, BDNF increased D1 receptor mRNA in a time‐ and dose‐dependent manner with a fourfold increase in D1 receptor mRNA observed as early as 3 h with 10 ng/mL of BDNF. Using different classes and concentrations of kinase inhibitors, we determined that BDNF‐induced increase of D1 receptor mRNA is mediated by the phosphatidylinositol 3‐kinase signaling pathway. The increase required both new transcription and protein synthesis, as it was blocked by actinomycin D and cyclohexamide, respectively. Promoter deletion analysis identified a D1 promoter region necessary for mediating the effect of BDNF. These results provide novel evidence that D1 dopamine receptor expression is regulated by BDNF and its signaling pathway.

Gonadotropin-Releasing Hormone-Stimulated Sperm Binding to the Human Zona Is Mediated by a Calcium Influx

Abstract The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca2+-free medium and in the presence of Ca2+ channel antagonists. We also examined the GnRH effect on the intracellular free Ca2+ concentration ([Ca2+]i). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca2+ was present in the medium. In Ca2+-free medium or in the presence of 400 nM nifedipine, 80 μM diltiazem, or 50 μM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca2+]i in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca2+]i was fast and transient, from a basal [Ca2+]i of 413 ± 22 nM to a peak value of 797 ± 24 nM. The GnRH-induced increase in [Ca2+]i was entirely due to a Ca2+ influx from the extracellular medium because the increase in [Ca2+]i was blocked by the Ca2+ chelator EGTA and by the Ca2+ channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca2+ influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca2+ influx but inhibited the progesterone-activated Ca2+ influx. Finally, the GnRH-induced Ca2+ influx was blocked by two specific GnRH antagonists, Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH and Ac-3,4-dehydro-Pro1,-p-fluoro-D-Phe2, D-Trp3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca2+]i through T-type, voltage-operated calcium channels.

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

Tubular overexpression of Gremlin in transgenic mice aggravates renal damage in diabetic nephropathy

Diabetic nephropathy (DN) is currently a leading cause of end-stage renal failure worldwide. Gremlin was identified as a gene differentially expressed in mesangial cells exposed to high glucose and in experimental diabetic kidneys. We have described that Gremlin is highly expressed in biopsies from patients with diabetic nephropathy, predominantly in areas of tubulointerstitial fibrosis. In streptozotocin (STZ)-induced experimental diabetes, Gremlin deletion using Grem1 heterozygous knockout mice or by gene silencing, ameliorates renal damage. To study the in vivo role of Gremlin in renal damage, we developed a diabetic model induced by STZ in transgenic (TG) mice expressing human Gremlin in proximal tubular epithelial cells. The albuminuria/creatinuria ratio, determined at week 20 after treatment, was significantly increased in diabetic mice but with no significant differences between transgenic (TG/STZ) and wild-type mice (WT/STZ). To assess the level of renal damage, kidney tissue was analyzed by light microscopy (periodic acid-Schiff and Masson staining), electron microscopy, and quantitative PCR. TG/STZ mice had significantly greater thickening of the glomerular basement membrane, increased mesangial matrix, and podocytopenia vs. WT/STZ. At the tubulointerstitial level, TG/STZ showed increased cell infiltration and mild interstitial fibrosis. In addition, we observed a decreased expression of podocin and overexpression of monocyte chemoattractant protein-1 and fibrotic-related markers, including transforming growth factor-β1, Col1a1, and α-smooth muscle actin. Together, these results show that TG mice overexpressing Gremlin in renal tubules develop greater glomerular and tubulointerstitial injury in response to diabetic-mediated damage and support the involvement of Gremlin in diabetic nephropathy.