Holly M. Brothers & Gary L. Wenk The role of neuroinflammation in the determination of regional and neuronal vulnerability in Alzheimer’s disease Holly M. Brothers & Gary L. Wenk Department of Psychology The Ohio State University Nothing to Disclose

Studies in our laboratory demonstrate that regional and cellular vulnerability in the brain are influenced by an interaction between: neuroinflammation, duration of inflammation, and gender. 1 1

What initiates neuroinflammation? What are its consequences? Microglial activation is an early event in the pathogenesis of AD that appears in brain regions that later show the greatest degree of atrophy. Mutant Proteins Synaptic Loss/ Neuronal Death Neuroinflammaiton is: Common to all neurodegenerative diseases Sufficient to cause dementia (dementia pugilistica) Epidemiology suggests that reducing neuroinflammaiton (NSAIDs) early in this process is protective Not sporatic, is initiated and perpetuated by mutant proteins associated with AD as well as cell death Most importantly, the distribution of activated immune cells is correlated with those regions that degenerate the most. A number of risk factors for AD have been identified, including age, ApoE4 genotype, Down’s syndrome, and history of head injury. A number of pathophysiologic abnormalities have also been identified in AD. The histopathologic hallmarks of AD include the deposition of -amyloid in senile plaques and blood vessel walls as well as the presence of neurofibrillary tangles in the brain. In addition, the levels of several neurotransmitters are greatly reduced. Depletion of ACh represents the most important event. The dramatic and global reduction of ACh and other important neurotransmitters is almost certainly responsible for the broad and profound clinical manifestations of AD (Samuels and Davis, 1997). Biochemical evidence suggests that inflammation and/or the deleterious effects of free radicals may also play a role in the pathogenesis of AD (Rogers, 1995; Rogers et al., 1992; Smith et al., 1991). Because oestrogen is important in the maintenance of cholinergic neurones, it is also believed that postmenopausal oestrogen deficiency may play a role in the disease (Gibbs and Aggarwal, 1998). The leading approaches to AD therapy today are based on cholinergic mechanisms. Future therapies to alter disease course will target neuro-fibrillary tangle formation and -amyloid metabolism. Dementia Symptoms Inflammation Cagnin et al., 2001

Chronic neuroinflammation model Infusion of lipopolysaccharide (LPS, 0.15µl/hr, 6µg/day) into the IVth ventricle or basal forebrain cannula tract LPS In order to study the effects of chronic neuroinflammaiton, we use a model created by Beatrice Hauss-Wzyneriac… Hippocampus is cradled between the third ventricle on the inside and the lateral ventricles. SN, unlike hippocampus, is not in direct contact with a ventricle. Lipopolysaccharide (LPS)- an endotoxin from the cell wall of gram-negative bacteria. Osmotic minipump- slow, consistent drug release.

Regional microglia activation LPS 21 days → activated microglia in the hippocampus and temporal lobe. 2 Days aCSF + Sal, 4 weeks 21 Days LPS + Sal, 4 weeks

Is there a pathological consequence? Fewer cresyl violet stained cells counted by stereology in entorhinal cortex layers II and III of rats infused with LPS for 37 days 1 2 3 4 5 6 7 8 9 * Total Number of Neurons (x104) aCSF LPS Cresyl violet staining of neurons, Does the paper say ‘large’ or define size anywhere? Hauss-Wegrzyniak et al., 2002

Is there a pathological consequence? Volumetric MRI analysis revealed that 42 days of LPS infusion caused a significant shrinkage of the temporal lobes and hippocampus and increase in the volume of the lateral ventricles. aCSF LPS T2-weighed MRI images Hippo and EC outlined in white (outlined in orange hidden behind). Hauss-Wegrzyniak et al., 2000

Is there a functional consequence? Impaired LTP induction in vivo in dentate gyrus of rats infused with LPS for 37 days Hauss-Wegrzyniak et al., 2002

What underlies a loss of LTP? Glutamate is necessary for LTP Glutamate dysfunction disrupts LTP Glutamate → NMDAR activation → Ca++ entry → Arc induction Mg+ Glutamatergic neuron Ca++ Arc 5 min 30 min Under normal circumstances, when learning occurs, only about 2% of the neurons in the hippocampus are activated – this is called “sparse encoding.” Arc in DG Rosi et al., 2005

Chronic neuroinflammation activates microglia (blue) and over-activates Arc (red) a-CSF Arc + hippocampal neurons: aCSF: ~2% LPS: ~4% LPS Double immunostaining for Arc protein (red) in granule cells and OX-6 protein (activated microglia, blue) in granule cells and hilus in the DG. (green marks nuclei and yellow results the nuclei of Arc positive cells) Evidence of excessive Ca++ entry and supports idea of excessive glutamate Rosi et al., 2005

What other regions are vulnerable to chronic neuroinflammation? LPS infused into the nucleus basalis magnocellularis (14, 37, 74 or 112 days) produced a time-dependent, but not dose-dependent, decline in the number of Choline acetyltransferase (ChAT)-immunoreactive cells. In frontal Cortex??? Willard et al., 1999

SN dopaminergic neurons are also vulnerable to chronic inflammation LPS + Sal aCSF + Sal a 7x basal levels of resting microglia demonstrates a link between microglia activation and vulnerable regions, depending on disease state

Does Gender influence the response to neuroinflammation? Female rats infused 42 days with LPS have ~1/2 the number of activated microglia as males. Estrogen replacement did not protect. ‡ 1600 ‡ 1400 1200 1000 + # Figure 5. Semiquantitative determination of the number of OX-6-positive microglia in the hippocampus, amygdala, lateral hypothalamus, zona incerta, central thalamic nucleus, cingulate gyrus/retrosplenial cortex, piriform/ entorhinal cortex, and parafascicular cortex was made by using four comparable regions from each rat from each group. The boundaries of the regions were defined with reference to an atlas of the rat brain (Pellegrino et al., 1979) and are shown in detail in Figure 1. Chronic lipopolysaccharide (LPS) activated microglia, which was not reduced by estrogen (E2; a mixture of 25% 17-estradiol and 75% cholesterol) replacement therapy. Intact females receiving LPS infusion had significantly fewer activated microglia. Oil was 100% cholesterol as vehicle. CSF cerebrospinal fluid infusion. Asterisks indicate a significant difference compared with all CSF-infused groups ( p .001); plus sign indicates a significant difference compared with the LPS intact group ( p .001); pound sign indicates a significant difference compared with LPS-infused ovariectomized rats ( p .02). Number of Activated Microglia 800 600 400 200 aCSF LPS aCSF LPS aCSF LPS Intact OVX OVX + Estrogen Marriott et al., 2002

Neuroinflammation did not impair the performance of intact females Microglia activation correlates with impaired water maze performance (r = 0.481, p<0.03) Neuroinflammation did not impair the performance of intact females 1200 1000 800 Path Length (cm) 600 Figure 2. Performance of young female rats in the Morris water maze task. In the absence of ovaries, chronic neuroinflammation impaired performance (** p .002) and chronic estrogen therapy impaired performance (* p .001), as compared with intact rats or CSF oil-treated rats. The combined presence of these two conditions produced a significantly greater impairment ( p .001) than either condition alone (i.e., as compared with the CSF E2 and LPS oil groups). Oil was 100% cholesterol as vehicle. CSF cerebrospinal fluid infusion; LPS lipopolysaccharide infusion; E2 estrogen (a mixture of 25% 17-estradiol and 75% cholesterol) treatment. aCSF OVX 400 LPS aCSF 200 Mean + SEM 1 2 3 4 Day of Testing Marriott et al., 2002

* In the absence of ovaries: Chronic neuroinflammation impaired performance. Chronic estrogen impaired performance. Together, ovariectomy and LPS were synergistic. 1200 1000 + LPS OVX + E2 LPS OVX aCSF OVX + E2 aCSF OVX LPS aCSF 800 Path Length (cm) * 600 Microglia activation correlates with impaired performance (r = 0.481, p<0.03) Neuroinflammation did not impair the performance of intact females In the absence of ovaries: Chronic neuroinflammation impaired performance. Chronic estrogen impaired performance. Together, ovariectomy and LPS were synergistic. Figure 2. Performance of young female rats in the Morris water maze task. In the absence of ovaries, chronic neuroinflammation impaired performance (** p .002) and chronic estrogen therapy impaired performance (* p .001), as compared with intact rats or CSF oil-treated rats. The combined presence of these two conditions produced a significantly greater impairment ( p .001) than either condition alone (i.e., as compared with the CSF E2 and LPS oil groups). Oil was 100% cholesterol as vehicle. CSF cerebrospinal fluid infusion; LPS lipopolysaccharide infusion; E2 estrogen (a mixture of 25% 17-estradiol and 75% cholesterol) treatment. 400 200 Mean + SEM 1 2 3 4 Day of Testing Marriott et al., 2002

Regional and cellular vulnerability is influenced by neuroinflammation, time and gender. Distribution of activated microglia changes over time and concentrates within regions prone to neurodegeneration. The loss of forebrain acetylcholine neurons and is time-dependent. Chronic neuroinflammation is likely responsible for the selective vulnerability of specific neural systems and brain regions associated with age-related neurodegenerative changes. Female rats show no neuroinflammation-induced cognitive impairment unless ovarian function is lost. The pattern of activated microglia evolves over time, from being uniformly distributed during the first few days to being concentrated within temporal lobe and basal forebrain regions after four weeks of LPS infusion. The loss of forebrain acetylcholine neurons is time-dependent. In contrast to males, female rats demonstrate a unique pattern of microglia activation and show no cognitive impairment in the presence of chronic neuroinflammation. The loss of ovarian function impairs performance in females with neuroinflammation. Chronic neuroinflammation is responsible for the selective vulnerability of specific neural systems and brain regions associated with age-related neurodegenerative changes.

Dr. Beatrice Hauss-Wegrzyniak Dr. Gary L. Wenk Dr. Beatrice Hauss-Wegrzyniak Dr. Susanna Rosi ICAD 2009 Dr. Lisa Marriott Dr. Yannick Marchalant Holly M. Brothers Supported by NIA Contact: Brothers.23@osu.edu