Faculty Bibliography

Down syndrome (DS) is caused by the triplication of human chromosome 21 (HSA21) and is the most common genetic cause of intellectual disability, with individuals having deficits in cognitive function including hippocampal learning and memory and neurodegeneration of cholinergic basal forebrain neurons, a pathological hallmark of Alzheimer's disease (AD). To date, the molecular underpinnings driving this pathology have not been elucidated. The Ts65Dn mouse is a segmental trisomy model of DS and like DS/AD pathology, displays age-related cognitive dysfunction and basal forebrain cholinergic neuron (BFCN) degeneration. To determine molecular and cellular changes important for elucidating mechanisms of neurodegeneration in DS/AD pathology, expression profiling studies were performed. Molecular fingerprinting of homogeneous populations of Cornu Ammonis 1 (CA1) pyramidal neurons was performed via laser capture microdissection followed by Terminal Continuation RNA amplification combined with custom-designed microarray analysis and subsequent validation of individual transcripts by qPCR and protein analysis via immunoblotting. Significant alterations were observed within CA1 pyramidal neurons of aged Ts65Dn mice compared to normal disomic (2N) littermates, notably in excitatory and inhibitory neurotransmission receptor families and neurotrophins, including brain-derived neurotrophic factor as well as several cognate neurotrophin receptors. Examining gene and protein expression levels after the onset of BFCN degeneration elucidated transcriptional and translational changes in neurons within a vulnerable circuit that may cause the AD-like pathology seen in DS as these individuals age, and provide rational targets for therapeutic interventions.

Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (alpha-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.

In catamenial epilepsy, seizures exhibit a cyclic pattern that parallels the menstrual cycle. Many studies suggest that catamenial seizures are caused by fluctuations in gonadal hormones during the menstrual cycle, but this has been difficult to study in rodent models of epilepsy because the ovarian cycle in rodents, called the estrous cycle, is disrupted by severe seizures. Thus, when epilepsy is severe, estrous cycles become irregular or stop. Therefore, we modified kainic acid (KA)- and pilocarpine-induced status epilepticus (SE) models of epilepsy so that seizures were rare for the first months after SE, and conducted video-EEG during this time. The results showed that interictal spikes (IIS) occurred intermittently. All rats with regular 4-day estrous cycles had IIS that waxed and waned with the estrous cycle. The association between the estrous cycle and IIS was strong: if the estrous cycles became irregular transiently, IIS frequency also became irregular, and when the estrous cycle resumed its 4-day pattern, IIS frequency did also. Furthermore, when rats were ovariectomized, or males were recorded, IIS frequency did not show a 4-day pattern. Systemic administration of an estrogen receptor antagonist stopped the estrous cycle transiently, accompanied by transient irregularity of the IIS pattern. Eventually all animals developed severe, frequent seizures and at that time both the estrous cycle and the IIS became irregular. We conclude that the estrous cycle entrains IIS in the modified KA and pilocarpine SE models of epilepsy. The data suggest that the ovarian cycle influences more aspects of epilepsy than seizure susceptibility.

Background: The risk of developing Alzheimer's disease (AD) is magnified in individuals with metabolic dysfunction, specifically obesity and type 2 diabetes. In cases of insulin deficiency or resistance, elevated and fluctuating levels of blood glucose lead to the production of Advanced Glycation End Products (AGEs) that bind their receptor, RAGE, with pathological consequences. AGE-RAGE ligand binding induces intracellular signaling cascades, in part via the formin signal transduction effector, diaphanous- 1 (mDia1). Activation of this cellular mechanism activates NF-KB activation, upregulates pro-inflammatory molecules, increases RAGE expression, and ignites a positive feedback loop driving chronic inflammation in the periphery. Given that AGEs are increased in the brains of both diabetic and AD patients, here we investigate AGE-RAGE binding and subsequent mDia1 signal transduction as a possible mechanism of neuroinflammation, which contributes to the pathogenesis of AD. Methods: mDia1 and RAGE expression in microglia was evaluated by Immunoflourescent IHC in temporal cortex brain slices of AD and Non-Demented Aged human tissue. CD11b+ microglia were isolated from young (<10 month) and old (>16 month) APP London mice and aged matched controls and subjected to molecular analysis. BV-2 microglial- like cells were stimulated by the prototypic RAGE ligand, Carboxy Methyl Lysine (CML-AGE) (100 mug/mL) for 24 h, harvested, and Ager (RAGE), Drf1 (mDia1), Cd36, and Cd68RNA transcript levels were analyzed by q-RT-PCR. Results: mDia1 and RAGE are highly expressed in human AD and aged brain and colocalize, at least in part, to CD68+ activated microglia. mDia1 is highly expressed in CD11b+ microglia from APP London mice vs. aged matched controls. CML-AGE stimulated BV-2 cells display a 2-fold increased expression of mDia1, RAGE, CD36, and CD68 vs. vehicle treatment. Conclusions: RAGE and mDia1 are highly expressed in AD human brain, supporting their possible role in mediating pathogenesis. Acute stimulation with RAGE ligands upregulates inflammatory and phagocytosis markers in cultured BV-2 cells. Primary isolates of murine CD11b+ microglia demonstrate high levels of expression of mDia. These data suggest that RAGE may exert its pathogenic effects in AD brain, at least in part via mDia1-mediated neuroinflammation, thereby driving the AD phenotype. (Figure presented)

Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders.

Endogenous murine amyloid-beta peptide (Abeta) is expressed in most Abeta precursor protein (APP) transgenic mouse models of Alzheimer's disease but its contribution to beta-amyloidosis remains unclear. We demonstrate approximately 35% increased cerebral Abeta load in APP23 transgenic mice compared with age-matched APP23 mice on an App-null background. No such difference was found for the much faster Abeta-depositing APPPS1 transgenic mouse model between animals with or without the murine App gene. Nevertheless, both APP23 and APPPS1 mice codeposited murine Abeta, and immunoelectron microscopy revealed a tight association of murine Abeta with human Abeta fibrils. Deposition of murine Abeta was considerably less efficient compared with the deposition of human Abeta indicating a lower amyloidogenic potential of murine Abeta in vivo. The amyloid dyes Pittsburgh Compound-B and pentamer formyl thiophene acetic acid did not differentiate between amyloid deposits consisting of human Abeta and deposits of mixed human-murine Abeta. Our data demonstrate a differential effect of murine Abeta on human Abeta deposition in different APP transgenic mice. The mechanistically complex interaction of human and mouse Abeta may affect pathogenesis of the models and should be considered when models are used for translational preclinical studies.

In the United States, estimates indicate there are between 250,000 and 400,000 individuals with Down syndrome (DS), and nearly all will develop Alzheimer's disease (AD) pathology starting in their 30s. With the current lifespan being 55 to 60 years, approximately 70% will develop dementia, and if their life expectancy continues to increase, the number of individuals developing AD will concomitantly increase. Pathogenic and mechanistic links between DS and Alzheimer's prompted the Alzheimer's Association to partner with the Linda Crnic Institute for Down Syndrome and the Global Down Syndrome Foundation at a workshop of AD and DS experts to discuss similarities and differences, challenges, and future directions for this field. The workshop articulated a set of research priorities: (1) target identification and drug development, (2) clinical and pathological staging, (3) cognitive assessment and clinical trials, and (4) partnerships and collaborations with the ultimate goal to deliver effective disease-modifying treatments.

Accumulating evidence shows that brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) significantly decrease early in Alzheimer's disease (AD). However, it remains unclear whether BDNF/TrkB reductions may be mechanistically involved in the pathogenesis of AD. To address this question, we generated 5XFAD transgenic mice with heterozygous TrkB knockout (TrkB(+/-).5XFAD), and tested the effects of TrkB reduction on AD-like features in this mouse model during an incipient stage that shows only modest amyloid-beta (Abeta) pathology and retains normal mnemonic function. TrkB(+/-) reduction exacerbated memory declines in 5XFAD mice at 4-5 months of age as assessed by the hippocampus-dependent spontaneous alternation Y-maze task, while the memory performance was not affected in TrkB(+/-) mice. Meanwhile, TrkB(+/-).5XFAD mice were normal in nest building, a widely used measure for social behavior, suggesting the memory-specific aggravation of AD-associated behavioral impairments. We found no difference between TrkB(+/-).5XFAD and 5XFAD control mice in cerebral plaque loads, Abeta concentrations including total Abeta42 and soluble oligomers and beta-amyloidogenic processing of amyloid precursor protein. Interestingly, reductions in hippocampal expression of AMPA/NMDA glutamate receptor subunits as well as impaired signaling pathways downstream to TrkB such as CREB (cAMP response element-binding protein) and Akt/GSK-3beta (glycogen synthase kinase-3beta) were observed in TrkB(+/-).5XFAD mice but not in 5XFAD mice. Among these signaling aberrations, only Akt/GSK-3beta dysfunction occurred in TrkB(+/-) mice, while others were synergistic consequences between TrkB reduction and subthreshold levels of Abeta in TrkB(+/-).5XFAD mice. Collectively, our results indicate that reduced TrkB does not affect beta-amyloidosis but exacerbates the manifestation of hippocampal mnemonic and signaling dysfunctions in early AD.