Infectious Agents in Neurodegenerative Disease

Infectious Agents in Neurodegenerative Disease 125

Infectious Agents in Neurodegenerative Disease M J Bellizzi and H A Gelbard, University of Rochester Medical Center, Rochester, NY, USA ã 2009 Elsevier Ltd. All rights reserved.

Introduction Neuronal apoptosis has been recognized for more than 10 years as one of the pathologic hallmarks of neurodegenerative disease associated with HIV-1 infection of the central nervous system (CNS), but mounting evidence suggests it has little to do with symptoms of neurologic disease associated with neurodegeneration. Studies of HIV-1-associated dementia (HAD) suggest that neurologic and cognitive function becomes impaired primarily as a result of injury to dendrites and synapses, often occurring long before cell death. HAD appears to develop not from loss of neurons but because of loss of communication between neurons and perhaps glia as well. This distinction has important implications for disease progression and treatment; dead neurons are not likely replaceable, except perhaps in areas of the CNS that may benefit from neurogenesis, but dendrites and synapses can recover from severe structural and functional injury as long as the neuronal cell body survives. With synaptic injury and clinical symptoms often preceding the extensive neuronal loss of latestage disease by months or years, therapeutic strategies that provide synaptic protection have potential to improve neurologic function and perhaps reverse the course of the disease. Dramatic recovery of neurologic function in patients with HAD beginning antiretroviral therapy demonstrates that a temporal window for reversibility of neurologic deficits does exist. HAD develops following HIV-1 infection of brain macrophages and microglia, and cognitive and motor symptoms include impaired concentration, apathy, memory loss, psychomotor slowing, weakness, tremor, and ataxia. These symptoms can be severely debilitating, but antiretroviral therapy that decreases HIV-1 burden in the brain and the periphery can ameliorate and even reverse many of these symptoms, at least temporarily. For example, an early report described a patient diagnosed with HIV-1 infection and HAD after presenting with 6 months of progressive memory and attentional deficits that had become so severe that she could no longer work, dress herself, or be left unsupervised. After 12 weeks of antiretroviral therapy that decreased viral burden and associated inflammatory markers in the plasma and cerebrospinal fluid, her performance on

cognitive and motor testing had dramatically improved and most, although not all, her presenting deficits were reversed. Similar clinical recovery, accompanied by significant improvement of cerebral atrophy documented by neuroimaging, was reported after antiretroviral therapy in a HIV-1-infected child with language, attention, and motor deficits that had caused him to lose developmental milestones and prevented him from communicating with others at school and at home. Studies following cohorts of patients beginning highly active antiretroviral therapy (HAART) provide further evidence of a large reversible component in HAD. HAART modifies the disease course but does not cure or prevent HAD. The prevalence of HAD and other neurologic disease in patients receiving HAART is the same as in the pre-HAART era. HAART has changed the phenotype of the disease, causing it to develop more slowly and shifting the pattern of clinical deficits, perhaps reflecting involvement of different brain areas: functional neuroimaging studies in the HAART era suggest substantial cortical involvement in a disease once considered to be primarily subcortical. The persistent 30–40% prevalence of HAD in HIV-positive cohorts despite HAART indicates that therapies targeting mechanisms that are independent of the viral life cycle with the goal of preventing synaptic injury are likely necessary for long-term treatment of HIV-1-associated neurologic disease. How HIV-1 damages synaptic architecture and function is not known, and understanding mechanisms of synaptic injury in HIV-1 neurologic disease is necessary for identifying therapeutic approaches to protect synaptic function. Similarities in disease pathology and neurotoxic mechanisms suggest that this understanding might provide insight into treatment for other neurodegenerative disorders with an infectious etiology.

HIV-1-Associated Dementia and Prionoses Are Diseases of Synaptic Architecture Evidence that neurologic impairment in HAD is caused by synaptic injury comes largely from correlations of neuropathologic studies with neurologic and neuropsychological testing. HIV-1 infection is associated with neuronal apoptosis, dramatic decreases in numbers of presynaptic terminals and postsynaptic dendritic spines, dendritic injury, and signs of HIV encephalitis, which include HIV-1-infected monocyte– macrophages, multinucleated giant cells, activated

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microglia, astrocytosis, and myelin pallor. Only synaptic loss and microglial activation correlate with the presence of neurologic deficits. While the multinucleated giant cells and myelin pallor of HIV encephalitis were seen only in patients with dementia, 50% of patients developed HAD in the absence of either of these findings. Similarly, premortem neurocognitive function did not correlate with the presence of microglial nodules, astrocytosis, or HIV-infected mononuclear cells in autopsy tissue. In contrast, loss of pre- and postsynaptic structures correlates well with neurologic impairment across a range of severity of HIV-1 neurologic disease. Studies of patients with HAD and milder forms of HIV-1associated neurologic impairment, as well as unimpaired HIV-1-seropositive controls, found that global ratings of neuropsychologic function derived from an extensive battery of tests significantly correlated with synaptic density estimated by presynaptic synaptophysin immunoreactivity as well as with postsynaptic microtubule-associated protein 2-positive dendritic area. Loss of synapses was not simply a result of neuronal death; neuronal loss consistently failed to correlate with neurologic impairment in a number of studies. While neuronal death clearly has an impact on neurologic function, these studies suggested that neuronal loss occurs late in the disease and that synaptic injury prior to neuronal death is the major determinant of HIV-associated neurologic disease. This is supported by morphologic studies that show extensive injury to the dendritic arbor, including focal swelling, or beading; loss of dendritic branches; and 40–60% reductions in densities of dendritic spines along remaining branches, all occurring on surviving neurons in HAD. The additional correlation between neurologic impairment and microglial activation, a general marker of neuroinflammation, suggests that inflammatory processes might contribute to synaptic injury. Similar evidence suggests that synaptic injury underlies neurologic deficits in other neurodegenerative diseases including the prionoses. Loss of synapses preceding neurodegeneration has been demonstrated in scrapie-infected murine hippocampus, with the major neuropathologic finding of abnormal pyramidal synapses in the scrapie-infected murine hippocampus. Loss of synapses and neurologic impairment correlates in the ME7 murine model of prion disease, and studies demonstrate similar synaptic pathology in patients with Creutzfeldt–Jakob disease (CJD) at both a light microscopic and ultrastructural level. Indeed, one of the most striking ultrastructural findings in a study of the CJD prionoses was dark profiles or ‘tombstones’ of synaptic terminals. In addition to loss of synaptic structures, functional impairment of existing synapses might

further disrupt neural processing in HAD. In addition, electrophysiologic studies in brain slices prepared from several different animal models of HAD have revealed deficits in synaptic plasticity, often occurring early in the disease and before overt neuropathological changes. It is interesting that in the ME7 model of prionoses, abnormalities in synaptic plasticity, while temporally contiguous with deposition of protease-resistant protein, scrapie-type (PrPSc), occur before overt changes in behavior or frank neuropathologic hallmarks of the prionoses such as vacuolation are evident. While much attention has been focused on neuroimaging techniques, including diffusion weighted images and fluid-attenuated inversion recovery images for the diagnosis of the prionoses, no studies have characterized progression of neurologic deficits and related it to changes in functional neuroimaging parameters, probably because of the difficulty in diagnosing this class of diseases in the asymptomatic phase and the rapid, inexorable progression of the disease once neurologic symptoms are apparent. In contrast, functional studies in patients with HIV-1 are consistent with impairment of synaptic processing even when there is no overt sign of neuropsychologic or neurologic deficit. HIV-1-positive patients with varying degrees of neurologic impairment showed increased areas of brain activation on functional magnetic resonance imaging during attention and working memory tasks. During simpler tasks, brain activation increased but performance did not deteriorate, whereas during more-complex tasks, larger increases in brain activation correlated with decreased performance. This finding supports the hypothesis that patients with impaired processing recruited additional neural substrate, or cognitive reserve to complete the tasks. It is interesting that cognitively asymptomatic HIV-1positive patients recruited larger brain areas, in the same regions as patients with HAD, compared to HIV-1-seronegative controls, suggesting that the former might already have had impairment of neural processing which was compensated by increased recruitment of cognitive reserve. Electroencephalographic studies during an auditory processing task support this conclusion. Patients with HIV-associated neurologic disease had longer latencies of eventrelated potentials that reflect impaired frontal cortical processing. Latencies increased with more-severe neurologic deficits and were also significantly longer in cognitively intact HIV-seropositive patients compared than in seronegative controls, again suggesting subclinical functional impairment during the asymptomatic phase of the disease. Taken together, these diverse studies suggest that neural processing is impaired in preclinical stages of

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HAD and worsens throughout disease progression in tandem with the development of progressively more severe synaptic pathology. Neuronal cell death likely becomes significant in late-stage disease, well after the onset of neuropsychologic impairment. These findings strongly support the hypothesis that synaptic injury is the primary determinant of neurologic impairment in HAD and very possibly the substrate for early neurologic disease in the prionoses. Thus, understanding mechanisms of synaptic injury and dysfunction in these diseases might be crucial to preserving neurologic function.

Inflammation and Excitotoxic Synaptic Injury How synapses are injured in prionoses is not well understood. Evidence for excitotoxic damage to synapses and neuronal cell bodies in the prionoses is equivocal, arguably because of the difficulty in understanding how PrPSc interacts with different cellular moieties germane to glutamate-mediated neurotransmission, and the finding that mice with a targeted deletion of the normal cellular prion protein do not display abnormalities in the kinetics of g-aminobutyric acid and glutamate receptor-mediated currents. It is interesting that plasminogen, a pro-protease that may participate in glutamate-mediated excitotoxicity, selectively binds PrPSc. However, neuropathologic studies of expression of excitatory amino acid transporter-1 (EAAT-1) in brain macrophages and microglia in patients with prion disease were unable to establish a clear relationship between EAAT-1’s ability to clear extracellular glutamate and the extent of neuronal apoptosis or spongiform neuropathologic changes. In contrast, in vitro studies have revealed many potential neurotoxic mechanisms for the pathogenesis of HAD, but investigation of how these contribute to synaptic dysfunction and destruction in vivo has been hampered by the slow progression of synaptic injury in these diseases. Nevertheless, multiple lines of evidence suggest that synaptic damage in HAD is likely due to chronic neuroinflammation associated with mitochondrial dysfunction, disrupted intracellullar calcium homeostasis, and oxidative stress in vulnerable neurons that increase synaptic vulnerability to glutamate excitotoxicity. HIV-1 rarely, if ever, infects postmitotic neurons. It exerts its neurotoxic effects largely by triggering brain inflammation. By infecting brain macrophages, microglia, and to a lesser extent astrocytes and secondarily activating uninfected microglia and astrocytes, HIV-1 causes release of cytokine and other inflammatory mediators, including tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), interleukin-6,

arachidonic acid metabolites, platelet-activating factor (PAF), and complement proteins, in addition to secreted HIV-1 proteins. This inflammatory milieu is toxic to neurons in vitro, as demonstrated by experiments with cerebrospinal fluid (CSF) from patients with HAD and conditioned media from HIV-infected macrophages. In patients with HIV, brain inflammation appears to relate closely to cognitive dysfunction: severity of dementia correlated well with numbers of activated microglia and macrophages and with TNF-a mRNA expression in the brain, but not with numbers of HIV-1-infected cells, and decreases in CSF TNF-a protein levels coincided with neurologic recovery in a patient beginning HAART. Evidence from in vitro studies suggests that HAD inflammatory mediators damage neurons by making them vulnerable to Ca2þ-dependent glutamate excitotoxicity. Supernatants containing the soluble mediators released by HIV-1- or amyloid-b-activated macrophages and microglia can kill cultured neurons in a manner that depends on activation of N-methylD-aspartate (NMDA)-type glutamate receptors. Many factors may contribute: proinflammatory molecules PAF, TNF-a, and IL-1b, as well as amyloid-b and the secreted HIV proteins gp120 and Tat, each exert neurotoxic effects largely by promoting glutamate excitotoxicity. Several studies are consistent with a role for excitotoxicity in vivo: NMDA receptordependent neuronal loss and neurotransmitter depletion have been observed, respectively, in rats injected with amyloid-b and a retroviral (LP-BM5) murine model of HAD. It is intriguing that studies with the LP-BM5 murine leukemia retrovirus (MuLV) model demonstrate production, probably by molecular mimicry, of virally induced auto-antibodies to alpha-amino-3-hydroxy5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, which are responsible in part for the neurodegenerative disease in this model. Furthermore, NMDA receptor antagonists prevent decline in synaptic function in the severe combined immunodeficiency mouse models of HIV-1 encephalitis and HAD. Glutamate excitotoxicity is widely believed to depend on Ca2þ influx into neurons via ionotropic glutamate receptors and subsequent uptake by mitochondria, leading to mitochondrial depolarization and dysfunction, including free radical generation and dysregulation of intracellular Ca2þ homeostasis, which can culminate in neuronal injury or death. HIV mediators likely contribute to this process at multiple stages. HIV-1, gp120, TNF-a, and amyloid-b can all inhibit glutamate uptake by cultured astrocytes, leading to higher extracellular glutamate concentrations in the brain. TNF-a, which can have toxic or protective effects depending on which receptors it activates, likely augments AMPA receptor-mediated glutamate toxicity

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in neurons by increasing the membrane expression of Ca2þ-permeable AMPA receptors. PAF increases neuronal glutamate release and promotes coupling of NMDA receptor activation with production of nitric oxide (NO), which links Ca2þ influx to mitochondrial dysfunction. Amyloid-b and HIV proteins can increase NO production and can lead to mitochondrial dysfunction and free radical generation in cultured neurons. Signs of mitochondrial dysfunction and oxidative stress have been found in patients with HAD compared with cognitively normal controls. In addition, treatment of cultured neurons with CSF from HIV-infected patients disrupts intracellular Ca2þ homeostasis, with higher Ca2þ concentrations at baseline and dramatically increased Ca2þ responses to glutamatergic stimulation. Impaired uptake of extracellular glutamate and increased sensitivity of neurons to glutamate-triggered Ca2þ influx and its downstream sequelae provide a compelling basis for the proposal that glutamate excitotoxicity mediated by ionotropic glutamate receptors might be a final common pathway to neuronal injury in HAD. This has led to considerable interest in therapeutic interventions that target NMDA receptor activation. However, the sequence of events leading to excitotoxic injury in chronic neurodegenerative disease is far from clear and could have important implications for the ability of glutamate receptor antagonists to mitigate excitotoxic injury. Many investigators have speculated that NMDA receptors are tonically activated in these diseases by chronically elevated glutamate concentrations in the synaptic cleft due to impaired astrocytic uptake of glutamate and/or by partial depolarization of neuronal membranes, perhaps due to mitochondrial dysfunction and impaired energy metabolism, that eliminates the voltage-dependent Mg2þ blockade of NMDA receptors and leads to sustained Ca2þ influx. This scenario provides the rationale for the therapeutic use of memantine, an uncompetitive NMDA receptor antagonist that blocks tonic but not transient activation. Direct evidence for this scenario in vivo is scarce, but indirect support comes largely from efficacy of treatment with memantine in disease models and in patients. Memantine has been shown to improve synaptic function and learning, respectively, in mouse models of HAD, but its ability to ameliorate symptoms of HAD is unclear. One way of explaining this surprising conundrum is that at early disease stages, neurons and astrocytes are not sufficiently dysfunctional to cause the tonic NMDA receptor activation that is most amenable to memantine blockade. Alternatively, effects of HAD inflammatory mediators might promote synaptic injury following more physiologic glutamate release. Recent work has demonstrated that high-frequency

synaptic stimulation, sufficient to generate long-term potentiation (LTP) in hippocampal slices, can instead initiate calcium- and caspase-dependent dendritic injury, with failure of LTP in slices exposed to cPAF, a PAF analog resistant to catabolism by tissue acetylhydrolases. Patch clamp recordings from postsynaptic neurons show no change in duration or extent of glutamate receptor activation during stimulation in cPAF-treated or control slices, suggesting that similar patterns of glutamatergic activity might induce physiologic plasticity or synaptic injury depending on whether neuroinflammation is present. Because we found that dendritic injury could be prevented and LTP restored by preconditioning with a mitochondrial adenosine triphosphate-sensitive Kþ channel antagonist, and that blockade of Ca2þ influx or postsynaptic caspase activation prevented structural injury but failed to restore LTP to cPAF-treated slices, we have concluded that abnormal NMDA receptor activation might not be sufficient to protect against synaptic injury during neuroinflammation. Rather, we believe that chemical preconditioning might provide a promising strategy for preventing activity-dependent synaptic injury while preserving physiologic plasticity. To date, most preclinical studies of neurotoxic mechanisms in HAD have used neuronal death as their endpoint. Thus, it would not be surprising if these studies offered more insight into end-stage disease, where cell death is far more prominent, than into earlier stages, where injury is restricted to synapses. Thus, development of model systems to investigate perturbation of synaptic structure and function in the absence of neuronal death might be important for identifying therapeutic strategies to prevent synaptic degeneration before it occurs irreversibly.

Synaptic Degeneration and Recovery Neuropathologic findings make clear that synapses degenerate before neurons die, but this progression is not inevitable. Abundant experimental evidence demonstrates that neurotoxicity can originate in and can be confined to pre- and postsynaptic elements and that synaptic structure and function can largely recover after removal of the neurotoxic insult. Molecular events associated with cell death, including caspase activation, mitochondrial calcium uptake and depolarization, free radical generation, loss of membrane asymmetry, and membrane blebbing, can occur locally in synapses exposed to glutamate, amyloid-b, prion protein, staurosporine, or Fe2þ-induced oxidative stress. Similar events may occur in presynaptic terminals and axons following loss of postsynaptic targets. Apoptotic responses can be generated in the synapses and dendrites, independent of the cell body,

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as demonstrated by application of toxins to isolated synaptic compartments or restricted portions of neurites. Though experiments with cytosolic extracts from synaptosomes show that synapse-derived signals can trigger apoptotic changes in cell nuclei, studies in intact neurons suggest that they remain localized to sites of excitotoxic exposure. Synapses and dendrites appear to be especially vulnerable to injury as cellwide exposures to sublethal doses of glutamate, amyloid-b, and prion protein can cause dendritic beading, caspase activation, and loss of membrane asymmetry in the dendrites while leaving the neuronal cell bodies relatively unaffected. Injured neurites have a remarkable capacity for recovery. In cultured neurons and hippocampal slices, sublethal doses of excitotoxins glutamate, kainate, or NMDA can dramatically disrupt dendritic architecture with large focal swellings and retraction of spines, accompanied by loss of synaptic transmission and changes in mitochondrial structure and function that can all recover following removal of the excitotoxin. Dendritic injury in these experiments reflects in part swelling due to ion influxes and osmotic changes, which can recover within minutes, and in part Ca2þmediated toxicity that can last from hours to days; nevertheless, dendrites can recover from both when the cell body is spared. Synaptic repair correlating with neurologic recovery has been observed in chronic neuroinflammatory disease models. In mice with experimental autoimmune encephalitis, a model for multiple sclerosis, beading of motor neuron dendrites and loss of immunostaining for pre- and postsynaptic markers accompanied episodes of motor dysfunction, and all returned to normal with remission of neurologic deficits. The remarkable regenerative potential of dendrites and synapses suggests a likely basis for the reversibility of HAD neuropsychologic symptoms in patients beginning HAART. Although such reversal has not been demonstrated in patients with other neurodegenerative diseases, or even in patients with HAD who have received long-term treatment with HAART, the potential for functional neurologic recovery in this patient group warrants further investigation with neuroprotective strategies that either reduce neuroinflammatory burden on synapses or precondition them to withstand this type of stress. This is even more compelling when one realizes that HAART fails to prevent HAD in the long run. Identifying potential targets for such therapies will require a better understanding, not of pathways to neuronal death, but of how HIV and inflammatory neurotoxins lead to synaptic injury and loss of function.

See also: Apoptosis in Neurodegenerative Disease; Excitotoxicity in Neurodegenerative Disease; Inflammation in Neurodegenerative Disease and Injury; Intracellular Calcium and Neuronal Death; Neural Repair and Regeneration: Inflammatory Mechanisms and Cytokines; Prion Diseases.

Further Reading Adle-Biassette H, Chretien F, Wingertsmann L, et al. (1999) Neuronal apoptosis does not correlate with dementia in HIV infection but is related to microglial activation and axonal damage. Neuropathology and Applied Neurobiology 25: 123–133. Anderson ER, Boyle J, Zink WE, Persidsky Y, Gendelman HE, and Xiong H (2003) Hippocampal synaptic dysfunction in a murine model of human immunodeficiency virus type 1 encephalitis. Neuroscience 118: 359–369. Bellichenko PV, Brown D, Jeffrey M, and Fraser JR (2000) Dendritic and synaptic alterations of hippocampal pyramidal neurons in scrapie-infected mice. Neuropathology and Applied Neurobiology 26: 143–149. Bellizzi MJ, Lu SM, Masliah E, and Gelbard HA (2005) Synaptic activity becomes excitotoxic in neurons exposed to elevated levels of platelet-activating factor. Journal of Clinical Investigation 115: 3185–3192. Chang L, Speck O, Miller EN, et al. (2001) Neural correlates of attention and working memory deficits in HIV patients. Neurology 57: 1001–1007. Chiti Z, Knutsen OM, Betmouni S, and Greene JR (2006) An integrated, temporal study of the behavioural, electrophysiological and neuropathological consequences of murine prion disease. Neurobiology of Disease 22(2): 363–373. Cunningham C, Deacon R, Wells H, et al. (2003) Synaptic changes characterize early behavioural signs in the ME7 model of murine prion disease. European Journal of Neuroscience 17: 2147–2155. Ellis R, Langford D, and Masliah E (2007) HIV and antiretroviral therapy in the brain: Neuronal injury and repair. Nature Reviews Neuroscience 8: 33–44. Ernst T, Chang L, Jovicich J, Ames N, and Arnold S (2002) Abnormal brain activation on functional MRI in cognitively asymptomatic HIV patients. Neurology 59: 1343–1349. Everall IP, Heaton RK, Marcotte TD, et al. (1999) Cortical synaptic density is reduced in mild to moderate human immunodeficiency virus neurocognitive disorder. Brain Pathology 9: 209–217. Gendelman HE, Zheng J, Coulter CL, et al. (1998) Suppression of inflammatory neurotoxins by highly active antiretroviral therapy in human immunodeficiency virus-associated dementia. Journal of Infectious Diseases 178: 1000–1007. Hasbani MJ, Hyrc KL, Faddis BT, Romano C, and Goldberg MP (1998) Distinct roles for sodium, chloride, and calcium in excitotoxic dendritic injury and recovery. Experimental Neurology 154: 241–258. Jeffrey M, Halliday WG, Bell J, et al. (2000) Synaptic loss associated with abnormal PrP precedes neuronal degeneration in the scrapie-infected murine hippocampus. Neuropathology and Applied Neurobiology 26: 41–54. Masliah E, Heaton RK, Marcotte TD, et al. (1997) Dendritic injury is a pathological substrate for human immunodeficiency virusrelated cognitive disorders. Annals of Neurology 42: 963–972. Mattson MP, Keller JN, and Begley JG (1998) Evidence for synaptic apoptosis. Experimental Neurology 153: 35–48.