Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia ~ 1997 International Society for Neurochemistry Prion Protein Fragment 106—126 Differentially Induces Heme Oxygenase- 1 mRNA in Cultured Neurons and Astroglial Cells M. Rizzardini, *R. Chiesa, *N. Angeretti, *E. Lucca, tM. Salmona, *G. Forloni, and L. Cantoni Heme and Hemoprotein Unit and Laboratories of *Biology of Neurodegenerative Disorders and ~Enzymology, Istituto di Ricerche Farmacologiche “Mario Negri,” Milan, Italy Abstract: Heme oxygenase (HO), which catalyzes the degradation of heme, has two isozymes (Ho-i and HO- 2). In brain the noninducible HO-2 isoform is predomi- nant, whereas the inducible HO-i is a marker of oxidative stress. Because brain oxidative stress might be present in prion-related encephalopathies (PREs), as in other neurodegenerative diseases, we investigated whether HO-i mANA was induced in neuronal and astroglial cell cultures by a peptide corresponding to residue i06—i26 of human prion protein (PrP). This peptide is amyloido- genic, and when added in vitro to cultured cells it repro- duces the neuronal death and astroglial proliferation and hypertrophy occurring in PREs. HO-i mRNA did not ac- cumulate in rat cultured neurons from hippocampus or cortex exposed to PrP 106—126 (50 p~M for 5 days). PrP 106—126 induced HO-i mRNA accumulation in rat astro- glial cultures depending on the exposure time and con- centration, being maximal (33-fold) after 7 days of expo- sure at 50 1iM. The nonamyloidogenic amidated or ami- dated-acetylated PrP i06—126 was ineffective, as was a scrambled peptide used as control. N-Acetylcysteine reduced (50%) the accumulation of HO-i mRNA in astro- glial cells after PrP i06—i26 (25 1sM) given for 5 days. Thus, oxidative stress is apparently a feature of the toxic- ity of PrP 106—126, and it might also occur in PRE5; induction of HO-i could contribute to the greater resis- tance of astrocytes compared with neurons to PrP i 06— i26 toxicity. Key Words: Heme oxygenase-i —Prion protein—Astroglial cells— Neurons—Oxidative stress— Prion-related encephalopathies. J. Neurochem. 68, 715—720 (i997). HO appears to exist as two isozymes: HO-i and HO-2. HO-2 is the constitutive form, and under normal conditions it is by far the predominant isozyme in the brain, where it is expressed in the cerebellum, fore- brain, brainstem, and diencephalon. HO-I under nor- mal conditions is detectable only in selected neuronal populations of the hippocampus and in the a-motor neurons in the spinal cord, the red nucleus, and dorsal raphe (Maines, 1993). However, in response to stress inducers there is a dramatic increase in HO-i protein in the choroidal lining of the ventricles of the brain as well as in glial cells throughout the organ including the cerebellum (Maines, 1993). Different stress stimuli rapidly induce HO-i (mRNA and protein) in brain in vivo, for example, hyperthermia, glutathione depletion, and reversible ischemia (Ewing et al., 1992; Ewing and Maines, 1993; Paschen et al., 1994). Heat shock or exposure to hydrogen peroxide has the same effect in cultured brain cells (Dwyer et al., i992, 1995). Apparently in other tissues too, HO-i is induced by various stimuli that have in common the ability to provoke oxidative stress (Applegate et al., 1991). A correlation was sometimes found between HO-i mRNA induction and cytoprotection (Nath et al., i 992); in cultured cortical neurons and astrocytes differential expression of HO- 1 is correlated to sensitivity to H2O2-induced injury (Dwyer et al., i995). Oxidative stress is implicated in several neurodegenerative diseases (Nixon and Ca- taldo, i994), and an increase in amount of HO-i pro- tein is associated with the neurofibrillary pathology of Alzheimer’ s disease (Premkumar et al., 1995; Schipper Heme oxygenase (EC 1.14.99.3; HO) catalyzes the rate-limiting step in heme degradation, yielding iron, biliverdin, and CO (Tenhunen et al., 1969); biliverdin is subsequently reduced enzymatically to bilirubin by biliverdin reductase. Both bilirubin and CO were sug- gested to have important physiological functions: bili- rubin as an antioxidant (Stocker et al., 1987) and CO as a messenger molecule (Marks et al., 1991; Verma et al., 1993). Received July 5, 1996; revised manuscript received October 2, 1996; accepted October 2, 1996. Address correspondence and reprint requests to Dr. L. Cantoni at Istituto di Ricerche Farmacologiche “Mario Negri,” Via Eritrea 62, 20157 Milan, Italy. Abbreviations used: Ac-PrP 106—126-NH2, amidated/acetylated prion protein 106—126; HO, heme oxygenase; NAC, N-acetylcys- teine; PRE, prion-related encephalopathy; PrP, prion protein; PrP 106—126-NH2, amidated prion protein 106—126; PrP~c, an isoform of prion protein that is protease-resistant. 715