Faculty Bibliography

Background: Dysfunction of the neuronal endosomal pathway is a characteristic of down syndrome (DS) and Alzheimer's disease (AD) and of carriers of the AD-risk apolipoprotein E 4 allele (APOE4). We hypothesized that the efficient release of endosomal material via exosomes into the extracellular space, as observed in the brains of DS patients and a mouse model of the disease and by DS fibroblasts, is necessary for a neuron to prevent accumulation of endosomal contents. Conversely, APOE4-driven downregulation of exosome release in the brains of APOE4 human carriers and APOE4 targeted-replacement mice appears to contribute to endosomal pathology. We investigated in vitro the interrelationship between the endosomal and exosomal pathways. Methods: Fibroblasts from DS patients and age-matched controls were transfected with CD63 siRNA or negative control siRNA. Level of exosomal secretion was studied by western blot analysis, and number and area of endosomes by immunohistochemistry. Results: Knockdown of the tetraspanin CD63, a regulator of exosome biogenesis, diminished exosome release by DS fibroblasts but not by control cells. CD63 knockdown did not affect endosomal morphology in control cells, but the number and total area occupied by endosomes was greater in DS fibroblasts in which CD63 expression was reduced. Summary/Conclusion: In neurodegenerative disorders with endosomallysosomal dysfunction, exosome secretion serves as a disposal mechanism for potentially toxic materials that are abnormally accumulated in endosomal compartments. Conversely, APOE4-driven downregulation of brain exosome biosynthesis and release contributes to endosomal pathology. Failure to maintain proper functioning of the interdependent endosomal-exosomal pathways during aging likely contributes to neuron degeneration and our findings argue that exosome production plays a central role maintaining homeostatic function of the endosomal-lysosomal system

Background: The apolipoprotein E (APOE) gene codes for the brain's primary cholesterol carrier protein. In both humans and humanized APOE mice the Alzheimer's disease-risk APOE 4 allele (APOE4) alters the number and size of neuronal endosomes, a pathology common to several neurodegenerative disorders, including Alzheimer's disease. Given that exosomes derive from the endosomal system, we investigated the impact of APOE4 on brain-derived exosomes. Methods: Extracellular vesicles (EV) were isolated from brain tissue of neuropathologically normal humans and of APOE targeted-replacement mice at 6, 12 and 18 months of age. Antibodies against TSG101 and ALIX were used to identify the exosome population within these samples. Protein, mRNA and lipid analyses were performed on both EV and whole-brain samples. Results: We found lower exosome levels in the brains of neuropathologically normal human APOE4 carriers compared to individuals homozygous for the risk-neutral 3 allele (APOE3). In APOE4 compared with APOE3 mice, brain exosome levels were lower in an age-dependent manner: lower levels were observed at 12 and 18 but not at 6 months of age. Protein and mRNA expressions of the exosome pathway regulators TSG101 and Rab35 were also lower in APOE4 compared with APOE3 mouse brains at 12 months of age, arguing for decreased exosome biosynthesis and secretion, respectively, from the endosomal pathway. Cholesterol and ganglioside levels were higher in brain exosomes isolated from 12-month-old APOE4 compared with APOE3 mice. Summary/Conclusion: Our findings show an APOE4-driven downregulation of brain exosome biosynthesis and release that is associated with altered lipid homeostasis. Failure to maintain proper functioning of the interdependent endosomal-exosomal pathways during aging, which is essential for diverse homeostatic and catabolic cellular processes, is likely to contribute to neuronal vulnerability in neurodegenerative disorders, including Alzheimer's disease

While apolipoprotein (Apo)E4 is linked to increased incidence of Alzheimer's disease (AD), there is growing evidence that it plays a role in functional brain irregularities that are independent of AD pathology. However, ApoE4-driven functional differences within olfactory processing regions have yet to be examined. Utilizing knock-in mice humanized to ApoE4 versus the more common ApoE3, we examined a simple olfactory perceptual memory that relies on the transfer of information from the olfactory bulb (OB) to the piriform cortex (PCX), the primary cortical region involved in higher order olfaction. In addition, we have recorded in vivo resting and odor-evoked local field potentials (LPF) from both brain regions and measured corresponding odor response magnitudes in anesthetized young (6-month-old) and middle-aged (12-month-old) ApoE mice. Young ApoE4 compared to ApoE3 mice exhibited a behavioral olfactory deficit coinciding with hyperactive odor-evoked response magnitudes within the OB that were not observed in older ApoE4 mice. Meanwhile, middle-aged ApoE4 compared to ApoE3 mice exhibited heightened response magnitudes in the PCX without a corresponding olfactory deficit, suggesting a shift with aging in ApoE4-driven effects from OB to PCX. Interestingly, the increased ApoE4-specific response in the PCX at middle-age was primarily due to a dampening of baseline spontaneous activity rather than an increase in evoked response power. Our findings indicate that early ApoE4-driven olfactory memory impairments and OB network abnormalities may be a precursor to later network dysfunction in the PCX, a region that not only is targeted early in AD, but may be selectively vulnerable to ApoE4 genotype.

Aims Biochemical and proteomic analysis of brain deposits and biological fluids reveal a high degree of Abeta heterogeneity that goes far beyond the classical Abeta40/Abeta42 dichotomy, displaying numerous post-translational modifications and multiple truncations at both N- and C-terminal ends of the molecule likely reflecting local action of resident enzymes. In spite of innumerable studies focusing in Abeta, the relevance of N- and C-terminal truncated species in the mechanism of AD pathogenesis remains largely understudied. Method Abeta species in brain tissue extracts were identified via immunoprecipitation/mass spectrometry. Synthetic homologues of intact and truncated peptides were compared in their solubility properties, self-oligomerization propensity, and brain clearance characteristics. Novel antibodies recognizing specific N- and C-terminal truncations were employed to immunolabel amyloid deposits in AD brains and transgenic models. Intracerebral injections of monomeric and oligomeric radiolabeled homologues were used to assess their brain clearance characteristics. Results N- and C-terminal truncated fragments in brain homogenates exhibit differential fractionation characteristics and topographic localization. Water-soluble brain extracts were enriched in C-terminal fragments -resembling the CSF Abeta peptidome- whereas N-terminal truncations required formic acid for solubilization. Synthetic homologues confirmed the differences in solubility and revealed contrasting oligomerization/ fibrillization characteristics. Notably, oligomerization largely increased brain retention, a characteristic mostly evident in fragments truncated at Phe4, topographically abundant in the plaque cores. Conclusion Abeta degradation at the C-terminal-end generates fragments likely associated to catabolic/clearance mechanisms while truncations at the N-terminus favor oligomerization and brain retention, with the potential to exacerbate the process of amyloidogenesis

Possession of the ε4 allele of apolipoprotein E (APOE) is the major genetic risk factor for late-onset Alzheimer's disease (AD). Although numerous hypotheses have been proposed, the precise cause of this increased AD risk is not yet known. In order to gain a more comprehensive understanding of APOE4's role in AD, we performed RNA-sequencing on an AD-vulnerable vs. an AD-resistant brain region from aged APOE targeted replacement mice. This transcriptomics analysis revealed a significant enrichment of genes involved in endosomal-lysosomal processing, suggesting an APOE4-specific endosomal-lysosomal pathway dysregulation in the brains of APOE4 mice. Further analysis revealed clear differences in the morphology of endosomal-lysosomal compartments, including an age-dependent increase in the number and size of early endosomes in APOE4 mice. These findings directly link the APOE4 genotype to endosomal-lysosomal dysregulation in an in vivo, AD pathology-free setting, which may play a causative role in the increased incidence of AD among APOE4 carriers.

Under normal conditions, the function of catalytically active proteases is regulated, in part, by their endogenous inhibitors, and any change in the synthesis and/or function of a protease or its endogenous inhibitors may result in inappropriate protease activity. Altered proteolysis as a result of an imbalance between active proteases and their endogenous inhibitors can occur during normal aging, and such changes have also been associated with multiple neuronal diseases, including Amyotrophic Lateral Sclerosis (ALS), rare heritable neurodegenerative disorders, ischemia, some forms of epilepsy, and Alzheimer's disease (AD). One of the most extensively studied endogenous inhibitor is the cysteine-protease inhibitor cystatin C (CysC). Changes in the expression and secretion of CysC in the brain have been described in various neurological disorders and in animal models of neurodegeneration, underscoring a role for CysC in these conditions. In the brain, multiple in vitro and in vivo findings have demonstrated that CysC plays protective roles via pathways that depend upon the inhibition of endosomal-lysosomal pathway cysteine proteases, such as cathepsin B (Cat B), via the induction of cellular autophagy, via the induction of cell proliferation, or via the inhibition of amyloid-beta (Abeta) aggregation. We review the data demonstrating the protective roles of CysC under conditions of neuronal challenge and the protective pathways induced by CysC under various conditions. Beyond highlighting the essential role that balanced proteolytic activity plays in supporting normal brain aging, these findings suggest that CysC is a therapeutic candidate that can potentially prevent brain damage and neurodegeneration.

Epidemiological findings suggest that diabetic individuals are at a greater risk for developing Alzheimer's disease (AD). To examine the mechanisms by which diabetes mellitus (DM) may contribute to AD pathology in humans, we examined brain tissue from streptozotocin-treated type 1 diabetic adult male vervet monkeys receiving twice-daily exogenous insulin injections for 8-20 weeks. We found greater inhibitory phosphorylation of insulin receptor substrate 1 in each brain region examined of the diabetic monkeys when compared with controls, consistent with a pattern of brain insulin resistance that is similar to that reported in the human AD brain. Additionally, a widespread increase in phosphorylated tau was seen, including brain areas vulnerable in AD, as well as relatively spared structures, such as the cerebellum. An increase in active ERK1/2 was also detected, consistent with DM leading to changes in tau-kinase activity broadly within the brain. In contrast to these widespread changes, we found an increase in soluble amyloid-beta (Abeta) levels that was restricted to the temporal lobe, with the greatest increase seen in the hippocampus. Consistent with this localized Abeta increase, a hippocampus-restricted decrease in the protein and mRNA for the Abeta-degrading enzyme neprilysin (NEP) was found, whereas various Abeta-clearing and -degrading proteins were unchanged. Thus, we document multiple biochemical changes in the insulin-controlled DM monkey brain that can link DM with the risk of developing AD, including dysregulation of the insulin-signaling pathway, changes in tau phosphorylation, and a decrease in NEP expression in the hippocampus that is coupled with a localized increase in Abeta. SIGNIFICANCE STATEMENT: Given that diabetes mellitus (DM) appears to increase the risk of developing Alzheimer's disease (AD), understanding the mechanisms by which DM promotes AD is important. We report that DM in a nonhuman primate brain leads to changes in the levels or posttranslational processing of proteins central to AD pathobiology, including tau, amyloid-beta (Abeta), and the Abeta-degrading protease neprilysin. Additional evidence from this model suggests that alterations in brain insulin signaling occurred that are reminiscent of insulin signaling pathway changes seen in human AD. Thus, in anin vivomodel highly relevant to humans, we show multiple alterations in the brain resulting from DM that are mechanistically linked to AD risk.

beta-amyloid precursor protein (APP) and amyloid beta peptide (Abeta) are strongly implicated in Alzheimer's disease (AD) pathogenesis, although recent evidence has linked APP-betaCTF generated by BACE1 (beta-APP cleaving enzyme 1) to the development of endocytic abnormalities and cholinergic neurodegeneration in early AD. We show that partial BACE1 genetic reduction prevents these AD-related pathological features in the Ts2 mouse model of Down syndrome. Partially reducing BACE1 by deleting one BACE1 allele blocked development of age-related endosome enlargement in the medial septal nucleus, cerebral cortex, and hippocampus and loss of choline acetyltransferase (ChAT)-positive medial septal nucleus neurons. BACE1 reduction normalized APP-betaCTF elevation but did not alter Abeta40 and Abeta42 peptide levels in brain, supporting a critical role in vivo for APP-betaCTF in the development of these abnormalities. Although ameliorative effects of BACE1 inhibition on beta-amyloidosis and synaptic proteins levels have been previously noted in AD mouse models, our results highlight the additional potential value of BACE1 modulation in therapeutic targeting of endocytic dysfunction and cholinergic neurodegeneration in Down syndrome and AD.

The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1exon8). We previously reported that PS1 L271V increased amyloid beta (Abeta) 42/40 ratios, while PS1exon8 reduced Abeta42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1exon8 did not rescue Abeta generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1exon8 is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1exon8 in vivo by crossing PS1exon8 transgenics with either PS1-null or Dutch APPE693Q mice. As a control, we crossed APPE693Q with mice expressing a deletion in an adjacent exon (PS1exon9). PS1exon8 did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1exon8 interacts with nicastrin, participating in the gamma-secretase complex formation. These data support that catalytically inactive PS1exon8 is generated physiologically and participates in protein-protein interactions.

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.