The secreted protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipid (LDL) receptor family members LDLR, very low density lipoprotein receptor (VLDLR) and apolipoprotein receptor 2 (ApoER2), and promotes their degradation in intracellular acidic compartments. In the liver, LDLR is a major controller of blood LDL levels, whereas VLDLR and ApoER2 in the brain mediate Reelin signaling, a critical pathway for proper development of the nervous system. Expression level of PCSK9 in the brain is highest in the cerebellum during perinatal development, but is also increased in the adult brain after ischemia. The mechanism of PCSK9 function and its involvement in neuronal apoptosis is poorly understood. We show here that RNAimediated knockdown of PCSK9 significantly reduced the death of potassium-deprived cerebellar granule neurons (CGN), as shown by reduced levels of nuclear phosphorylated c-Jun and activated caspase-3, as well as condensed apoptotic nuclei. ApoER2 protein levels were increased in PCSK9 RNAi cells. Knockdown of ApoER2 but not of VLDLR was sufficient to reverse the protection provided by PCSK9 RNAi, suggesting that proapoptotic signaling of PCSK9 is mediated by altered ApoER2 function. Pharmacological inhibition of signaling pathways associated with lipoprotein receptors suggested that PCSK9 regulates neuronal apoptosis independently of NMDA receptor function but in concert with ERK and JNK signaling pathways. PCSK9 RNAi also reduced staurosporineinduced CGN apoptosis and axonal degeneration in the nerve growth factor-deprived dorsal root ganglion neurons. We conclude that PCSK9 potentiates neuronal apoptosis via modulation of ApoER2 levels and related anti-apoptotic signaling pathways.

Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-ß (TGFß) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BREluciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3 H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.

Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant. Abbreviations: BMP15, Bone morphogenetic protein-15; BMPR2, BMP receptor 2; BS3, bis(sulfosuccinimidyl) suberate; DSS, disuccinimidyl suberate; DTT, dithiothreitol; ECD, ectodomain; GDF9B, growth differentiation factor-9B; hBMP15, human BMP15; hBMP15wt, hBMP15 wild type; hGL, human granulosa luteal; mAb-28, monoclonal antibody 28.

Disruption of the circadian rhythms is a frequent preclinical and clinical manifestation of Alzheimer’s disease. Furthermore, it has been suggested that shift work is a risk factor for Alzheimer’s disease. Previously, we have reported association of intolerance to shift work (job-related exhaustion in shift workers) with a variant rs12506228A, which is situated close to melatonin receptor type 1A gene (MTNR1A) and linked to MTNR1A brain expression levels. Here, we studied association of that variant with clinical and neuropathological Alzheimer’s disease in a Finnish whole-population cohort Vantaa 85+ (n = 512, participants over 85 years) and two follow-up cohorts. Rs12506228A was associated with clinical Alzheimer’s disease (p = 0.000073). Analysis of post-mortem brain tissues showed association with higher amount of neurofibrillary tangles (p = 0.0039) and amyloid beta plaques (p = 0.0041). We then followed up the associations in two independent replication samples. Replication for the association with clinical Alzheimer’s disease was detected in Kuopio 75+ (p = 0.012, n = 574), but not in the younger case-control sample (n = 651 + 669). While melatonin has been established in regulation of circadian rhythms, an independent role has been also shown for neuroprotection and specifically for anti-amyloidogenic effects. Indeed, in vitro, RNAi mediated silencing of MTNR1A increased the amyloidogenic processing of amyloid precursor protein (APP) in neurons, whereas overexpression decreased it. Our findings suggest variation close to MTNR1A as a shared genetic risk factor for intolerance to shift work and Alzheimer’s disease in old age. The genetic associations are likely to be mediated by differences in MTNR1A expression, which, in turn, modulate APP metabolism.