During development of the nervous system, as many as half of all neurons generated are ultimately eliminated by a process known as programmed cell death. Much of this cell death occurs as newly differentiated neurons compete for limiting amounts of survival-promoting 'neurotrophic' factors. Though counterintuitive, the selective death of neurons at specific times during development is critical for sculpting a properly wired nervous system. While programmed cell death is essential for normal development, too much or too little cell death later in life is a confounding factor in diseases ranging from Alzheimer's disease and stroke to brain cancer. Research in the Freeman laboratory is aimed at characterizing the mechanisms that regulate cell death in the mammalian nervous system. More specifically, we aim to identify and understand the critical cell signaling events that, if left unchecked, commit a neuron to die.

Our basic approach involves comparing gene expression and protein function in neurons before and after exposure to a death-inducing stimulus. For example, to study programmed cell death during development, we use a model in which neurons are deprived of the neurotrophic factor nerve growth factor. Using this model, we have discovered new roles during cell death for two proline-modifying enzymes, the prolyl hydroxylase EGLN3 and the peptidyl-prolyl isomerase PIN1. Using techniques and approaches from cell and molecular biology, genetics, and biochemistry, we are (1) determining the effects of knocking out these proteins on cell death during development and disease, (2) identifying their biochemical targets and substrates, and (3) characterizing the pathways that regulate their function in dying neurons.

A second interest of the laboratory concerns the mechanisms by which oxygen availability regulates the survival of developing neurons. Prenatal or perinatal hypoxia and hypoxia-ischemia are important causes of neonatal brain injury and abnormal brain development. To better understand these processes, we are investigating the regulation and function of the hypoxia-inducible factor (HIF) family of transcription factors in neurons exposed to different oxygen tensions. Ultimately, our research efforts are driven by the prospect that the mechanisms we uncover may ultimately contribute to the development of new therapies for cell death-related diseases and disorders of the nervous system.

• Fernandes KA, Harder JM, Fornarola LB, Freeman RS, Clark AF, Pang IH, John SW, Libby RT. JNK2 and JNK3 are major regulators of axonal injury-induced retinal ganglion cell death. Neurobiol Dis. 2012 May; 46(2):393-401.
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• Guo H, Barrett TM, Zhong Z, Fernández JA, Griffin JH, Freeman RS, Zlokovic BV. Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway. Mol Neurodegener. 2011; 6:13.
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• Zhong Z, Wang Y, Guo H, Sagare A, Fernández JA, Bell RD, Barrett TM, Griffin JH, Freeman RS, Zlokovic BV. Protein S protects neurons from excitotoxic injury by activating the TAM receptor Tyro3-phosphatidylinositol 3-kinase-Akt pathway through its sex hormone-binding globulin-like region. J Neurosci. 2010 Nov 17; 30(46):15521-34.
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• Xie L, Xiao K, Whalen EJ, Forrester MT, Freeman RS, Fong G, Gygi SP, Lefkowitz RJ, Stamler JS. Oxygen-regulated beta(2)-adrenergic receptor hydroxylation by EGLN3 and ubiquitylation by pVHL. Sci Signal. 2009; 2(78):ra33.
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• Guo Y, Schoell MC, Freeman RS. The von Hippel-Lindau protein sensitizes renal carcinoma cells to apoptotic stimuli through stabilization of BIM(EL). Oncogene. 2009 Apr 23; 28(16):1864-74.
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• Lomb DJ, Desouza LA, Franklin JL, Freeman RS. Prolyl hydroxylase inhibitors depend on extracellular glucose and hypoxia-inducible factor (HIF)-2alpha to inhibit cell death caused by nerve growth factor (NGF) deprivation: evidence that HIF-2alpha has a role in NGF-promoted survival of sympathetic neurons. Mol Pharmacol. 2009 May; 75(5):1198-209.
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• Ratan RR, Siddiq A, Aminova L, Langley B, McConoughey S, Karpisheva K, Lee HH, Carmichael T, Kornblum H, Coppola G, Geschwind DH, Hoke A, Smirnova N, Rink C, Roy S, Sen C, Beattie MS, Hart RP, Grumet M, Sun D, Freeman RS, Semenza GL, Gazaryan I. Small molecule activation of adaptive gene expression: tilorone or its analogs are novel potent activators of hypoxia inducible factor-1 that provide prophylaxis against stroke and spinal cord injury. Ann N Y Acad Sci. 2008 Dec; 1147:383-94.
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• Barone MC, Desouza LA, Freeman RS. Pin1 promotes cell death in NGF-dependent neurons through a mechanism requiring c-Jun activity. J Neurochem. 2008 Jul; 106(2):734-45.
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• Lomb DJ, Straub JA, Freeman RS. Prolyl hydroxylase inhibitors delay neuronal cell death caused by trophic factor deprivation. J Neurochem. 2007 Dec; 103(5):1897-906.
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• Fu J, Menzies K, Freeman RS, Taubman MB. EGLN3 prolyl hydroxylase regulates skeletal muscle differentiation and myogenin protein stability. J Biol Chem. 2007 Apr 27; 282(17):12410-8.
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• Brookes PS, Freeman RS, Barone MC. A shortcut to mitochondrial signaling and pathology: a commentary on "Nonenzymatic formation of succinate in mitochondria under oxidative stress". Free Radic Biol Med. 2006 Jul 1; 41(1):41-5.
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• Lee S, Nakamura E, Yang H, Wei W, Linggi MS, Sajan MP, Farese RV, Freeman RS, Carter BD, Kaelin WG, Schlisio S. Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer. Cancer Cell. 2005 Aug; 8(2):155-67.
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• Clifton DR, Rydkina E, Huyck H, Pryhuber G, Freeman RS, Silverman DJ, Sahni SK. Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after Rickettsia rickettsii infection: regulation by nuclear transcription factor NF-kappaB. Int J Med Microbiol. 2005 Aug; 295(4):267-78.
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• Xie L, Johnson RS, Freeman RS. Inhibition of NGF deprivation-induced death by low oxygen involves suppression of BIMEL and activation of HIF-1. J Cell Biol. 2005 Mar 14; 168(6):911-20.
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• Freeman RS, Barone MC. Targeting hypoxia-inducible factor (HIF) as a therapeutic strategy for CNS disorders. Curr Drug Targets CNS Neurol Disord. 2005 Feb; 4(1):85-92.
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• Clifton DR, Rydkina E, Freeman RS, Sahni SK. NF-kappaB activation during Rickettsia rickettsii infection of endothelial cells involves the activation of catalytic IkappaB kinases IKKalpha and IKKbeta and phosphorylation-proteolysis of the inhibitor protein IkappaBalpha. Infect Immun. 2005 Jan; 73(1):155-65.
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• Farhana L, Dawson MI, Huang Y, Zhang Y, Rishi AK, Reddy KB, Freeman RS, Fontana JA. Apoptosis signaling by the novel compound 3-Cl-AHPC involves increased EGFR proteolysis and accompanying decreased phosphatidylinositol 3-kinase and AKT kinase activities. Oncogene. 2004 Mar 11; 23(10):1874-84.
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• Freeman RS, Burch RL, Crowder RJ, Lomb DJ, Schoell MC, Straub JA, Xie L. NGF deprivation-induced gene expression: after ten years, where do we stand? Prog Brain Res. 2004; 146:111-26.
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• Besirli CG, Deckwerth TL, Crowder RJ, Freeman RS, Johnson EM. Cytosine arabinoside rapidly activates Bax-dependent apoptosis and a delayed Bax-independent death pathway in sympathetic neurons. Cell Death Differ. 2003 Sep; 10(9):1045-58.
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• Freeman RS, Hasbani DM, Lipscomb EA, Straub JA, Xie L. SM-20, EGL-9, and the EGLN family of hypoxia-inducible factor prolyl hydroxylases. Mol Cells. 2003 Aug 31; 16(1):1-12.
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• Straub JA, Lipscomb EA, Yoshida ES, Freeman RS. Induction of SM-20 in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death. J Neurochem. 2003 Apr; 85(2):318-28.
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• Wolf G, Harendza S, Schroeder R, Wenzel U, Zahner G, Butzmann U, Freeman RS, Stahl RA. Angiotensin II's antiproliferative effects mediated through AT2-receptors depend on down-regulation of SM-20. Lab Invest. 2002 Oct; 82(10):1305-17.
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• Sarmiere PD, Freeman RS. Analysis of the NF-kappa B and PI 3-kinase/Akt survival pathways in nerve growth factor-dependent neurons. Mol Cell Neurosci. 2001 Sep; 18(3):320-31.
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• Crowder RJ, Freeman RS. Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal. J Biol Chem. 2000 Nov 3; 275(44):34266-71.
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• Lipscomb EA, Sarmiere PD, Freeman RS. SM-20 is a novel mitochondrial protein that causes caspase-dependent cell death in nerve growth factor-dependent neurons. J Biol Chem. 2001 Feb 16; 276(7):5085-92.
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• Crowder RJ, Freeman RS. The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3-kinase and Akt. J Neurochem. 1999 Aug; 73(2):466-75.
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• Lipscomb EA, Sarmiere PD, Crowder RJ, Freeman RS. Expression of the SM-20 gene promotes death in nerve growth factor-dependent sympathetic neurons. J Neurochem. 1999 Jul; 73(1):429-32.
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• Maggirwar SB, Sarmiere PD, Dewhurst S, Freeman RS. Nerve growth factor-dependent activation of NF-kappaB contributes to survival of sympathetic neurons. J Neurosci. 1998 Dec 15; 18(24):10356-65.
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• Crowder RJ, Freeman RS. Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J Neurosci. 1998 Apr 15; 18(8):2933-43.
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• Estus S, Zaks WJ, Freeman RS, Gruda M, Bravo R, Johnson EM. Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis. J Cell Biol. 1994 Dec; 127(6 Pt 1):1717-27.
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• Freeman RS, Estus S, Johnson EM. Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of Cyclin D1 during programmed cell death. Neuron. 1994 Feb; 12(2):343-55.
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• Smith CJ, Johnson EM, Osborne P, Freeman RS, Neveu I, Brachet P. NGF deprivation and neuronal degeneration trigger altered beta-amyloid precursor protein gene expression in the rat superior cervical ganglia in vivo and in vitro. Brain Res Mol Brain Res. 1993 Mar; 17(3-4):328-34.
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• Freeman RS, Estus S, Horigome K, Johnson EM. Cell death genes in invertebrates and (maybe) vertebrates. Curr Opin Neurobiol. 1993 Feb; 3(1):25-31.
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• Freeman RS, Meyer AN, Li J, Donoghue DJ. Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor. J Cell Biol. 1992 Feb; 116(3):725-35.
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• Freeman RS, Donoghue DJ. Protein kinases and protooncogenes: biochemical regulators of the eukaryotic cell cycle. Biochemistry. 1991 Mar 5; 30(9):2293-302.
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• Freeman RS, Ballantyne SM, Donoghue DJ. Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mosXe. Mol Cell Biol. 1991 Mar; 11(3):1713-7.
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• Freeman RS, Kanki JP, Ballantyne SM, Pickham KM, Donoghue DJ. Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos. J Cell Biol. 1990 Aug; 111(2):533-41.
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• Freeman RS, Donoghue DJ. Transforming mutant v-mos protein kinases that are deficient in in vitro autophosphorylation. Mol Cell Biol. 1989 Sep; 9(9):4087-90.
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• Freeman RS, Pickham KM, Kanki JP, Lee BA, Pena SV, Donoghue DJ. Xenopus homolog of the mos protooncogene transforms mammalian fibroblasts and induces maturation of Xenopus oocytes. Proc Natl Acad Sci U S A. 1989 Aug; 86(15):5805-9.
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