Department of Medicine University of Alberta Edmonton Alberta

Department of Medicine University of Alberta Edmonton Alberta

Overview of Topics Impact of HAART on HIV neuroinvasion, neurotropism, neurovirulence. Emerging concepts derived from animal models, new technologies and recent studies in proteolytic activity Future perspectives

NEUROTROPIC RETROVIRUSES MuLV lentiviruses spumaviruses (HSRV) D-type (HERV-W-, SMRV) FIV EIAV HIV-1 CAEV VMV SIVagm HIV-2 SIVmac primate non-primate B-type (HERV-K, MMTV) ALV-like (RSV) HTLV-I/II LTR gag pol env LTR Power, Trends Neurosci (2001)

HIV-1 systemic disease course Infection Seroconversion Death Disease Progression Primary infection Asymptomatic AIDS Coreceptor use CCR5 CXCR4/CCR5 Molecular Distance from Founder Virus Viral/Host Variables CD4 + Lymphocytes Viremia 4-8 weeks 10 years 1-4 years

Primary HIV-induced neurological syndromes Seropositive Asymptomatic AIDS Acute meningitis Chronic meningitis GBS/CIDP Mononeuritis Neurocognitive impairment MCMD HAD Myelopathy Polyneuropathy DSP and ATN Myopathy

HIV neuropathology in the HAART era MGN HIV gp41 MNGC PVC

Depletion of HIV-1 p24-postive macrophages in brains of SCID mice by HAART Cook et al, Ann Neurol, 2005

AIDS-nondemented HIV Dementia MAP-2 p53 p53

HIV neuropathogenesis Astrocyte (activation/death) lymphocyte CD4 CCR5 Antibody response macrophage NO, QA Glutamate CXCR4 Neuroinvasion MMPs gp120, Tat Neurotropism neuron (injury/death) Neurovirulence macrophage CTL response Blood PVM Brain microglia Triggered Mechanisms: Inflammatory response Release of neurotoxic molecules Cell death/injury

Early brain injury in SIV-infected macaques Gonzalez et al, AIDS, 2000

HIV cell tropism in the CNS Gonzalez-Scarano and Martin-Garcia, Nat Rev Immunol, 2005

HIV-1 organ compartmentalization A subtype D subtype (B) (B) (M) (B) (B) (B) (B) (B) (M) (T) (T) (T) (T) (T) (T) B subtype Zhang et al, Virology 2001

Neurovirulence Mechanisms of neurodegeneration: Direct cytotoxic effects of viral proteins (gp120, gp41, Tat, Vpr) Indirect (bystander) effects of immune activation of brain monocytoid cells and/or astrocytes

Determinants of Neurovirulence Viral transcription Viral replication Neurotropism proviral DNA Apoptosis Apoptosis Neuroinflammation Neurotoxin induction LTR gag pol Vif Rev1 Vpu Vpr Tat1 env Rev2 Tat2 Nef LTR Tat Viral replication Neurotropism Viral variability Host transcription Neuroinflammation Apoptosis Apoptosis Neuroinflammation Neurotoxin induction

Direct effects of HIV proteins on neural cell viability and function Perruzi et al, BRRR, 2005

Indirect neurotoxicity mediated by supernatants from FIV-infected/activated macrophages % increase in neuronal death Health Disease Bragg et al, NBD, 2002

Activated microglia and macrophages: key players in brain innate immunity Normal immune sensors Activated phagocytosis chemotaxis antigen presentation cytotoxicity morphological changes proliferation respiratory burst MMPs, HSP proteases T CELL IFN- IL-1 ROS (NO) MMPs EAAs TNF Neuronal injury/death IL-1, IL-6, TNF MICROGLIA IL-1 IL-6 TNF ASTROCYTE microglia/ macrophage Complement components MHC II, adhesion molecules.

Astrocyte: the most abundant cell in the brain

Cell cycle genes perturbed by HIV infection of astrocytes Kim et al, JNI, 2004

CNS proteolytic activity: determinants of neuronal survival and death

Proteolytic cleavage of amyloid precursor protein (APP) I. APP 1 NH 2 II. Amyloidogenic cleavages BACE1 BACE1 671 A 770 COOH cytoplasmic -secretase EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQ 671 BACE1 -CTF -CTF C99 C89 -secretase A 1-40, 1-42 A 11-40, 11-42 Neurotoxicity

Detection of intracellular -amyloid in brains of AIDS patients Green et al, AIDS 2005

upar is upregulated in HAD Cinque et al, Ann Neurol, 2004

Proteinase-activated receptors (PARs) Hollenberg et al. Pharmacol Rev. 2002

Proteinase activated receptor-2 is up-regulated on neurons in HIV-induced neuroinflammation mrna RFC 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 HIV-ND HAD * TNF- TNF-a IL-1 IL-1b * mrna RFC 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 HIV-ND * HAD PAR-2 Trypsinogen HIV-ND HAD

PAR-2 deficiency exacerbates HIV Tat-induced neuropathology and neurobehavioral deficits A KO NeuN GFAP Merged image NeuN GFAP Merged image WT B 25 20 15 Reactivity/ Hpf 10 5 0 WT KO * NeuN * GFAP Percent of ipsiversive rotations 70 60 50 40 30 20 10 0 WT KO * Day 3 Day 7 Day 3 Day 7 Noorbakhsh et al, J. Immunol, 2005

Cleaved SDF-1 is highly neurotoxic and present in cortex of HAD patients SDF-1 SDF-1 SDF-1(5-67) Neuronal Death 8 6 4 SDF-1(5-67) Control 50 40 60 50 TUNEL Control SDF-1 ND HAD 40 Actin 2 0 0 SDF (1-67) 50 SDF (5-67) * ** ** 1 10 100 1000 Chemokine (nm)

Neuropathological consequences of SDF-1 implantation PBS SDF 1-67 SDF 5-67 Microglia A B C Astrocytes D E F Neurons G H I Zhang et al, Nat Neurosci (2003)

Proteolysis of SDF-1 results in neuronal death HIV-infected or activated macrophage/microglia pro MMP-2 MMPI activated MMP-2 C-SDF SDF-1 Cytokines, RR, gp120 and Tat p53 G-PCR Apoptotic neuron Activated astrocyte

Minocycline is neuroprotective and reduces viral replication in SIV infection Zink et al, JAMA, 2005

Growth hormone is neuroprotective Neuronal death RFI (±SD) 14 12 10 8 6 4 2 ** * *** *** NG108 LAN-2 *** *** % Ipsiversive rotations (±SD) 90 80 70 60 50 40 30 20 10 0 Day 7 * 0 Control Tat +GH 0.1 +GH 1.0 +GH 10 +GH 100 Control Tat Tat-GH 0.5 mg/kg Tat-GH 0.05 mg/kg In vitro In vivo Silva et al, Ann Neurol, 2003

Growth hormone protects neural stem cells in vivo in FIV infection Fold increase 12 10 8 6 4 2 * IDO Fold increase 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 Nestin * Fold increase 2.5 2 1.5 1 0.5 IGF-1 ** 0 FIV- FIV+ FIV+ &GH 0 FIV- FIV+ FIV+ &GH 0 FIV- FIV+ FIV+ &GH * P< 0.05

Growth hormone prevents FIV-induced immunosuppression and neurological deficits CD4 + cells 40 FIV+&GH % Cells ± SD 30 20 10 *** FIV+ FIV- 2 1.5 Neurobehavioral score (week 12) 0 Week 8 Week 12 MDS 1 ** CD8 + cells 0.5 50 40 *** 0 % Cells ± SD 30 20 10 FIV+&GH FIV+ FIV- FIV(-) FIV(+) FIV(+) &GH *** P< 0.001 ** P< 0.01 0 Week 8 Week 12 van Marle et al, submitted

Future Perspectives Drug-resistant virus and neurological disease Effects of dual/suprainfections (HIV, HCV, HERVs) on neuropathogenesis Mechanisms of neurological disease in HIV-1 non-b clade infections New non-arv therapies for HIV-related neurological disorders

Acknowledgements NEUROVIROLOGY LAB: Guido van Marle, Farshid Noorbakhsh, Kunyan Zhang, David Vergote, Neda Sharief, Claudia Silva, Yu Zhu, Shuhong Liu COLLABORATORS: John Gill/John Wallace/Morley Hollenberg (UCal), Justin McArthur/Carlos Pardo (JHU), Chris Overall (UBC) Funds: Alberta Heritage Foundation for Medical Research (AHFMR) Canadian Institutes for Health Research (CIHR) Canadian Foundation for AIDS Research (CANFAR) National Institutes of Health (NIH)