Faculty Bibliography

Dysfunction of the endosomal-lysosomal system is a prominent pathogenic factor in Alzheimer's disease (AD) and other neurodevelopmental and neurodegenerative disorders. We and others have extensively characterized the neuronal endosomal pathway pathology that results from either triplication of the amyloid-β precursor protein (APP) gene in Down syndrome (DS) or from expression of the apolipoprotein E ε4 allele (APOE4), the greatest genetic risk factor for late-onset AD. More recently brain exosomes, extracellular vesicles that are generated within and released from endosomal compartments, have been shown to be altered in DS and by APOE4 expression. In this review, we discuss the emerging data arguing for an interdependence between exosome production and endosomal pathway integrity in the brain. In vitro and in vivo studies indicate that altered trafficking through the endosomal pathway or compromised cargo turnover within lysosomes can affect the production, secretion, and content of exosomes. Conversely, exosome biogenesis can affect the endosomal-lysosomal system. Indeed, we propose that efficient exosome release helps to modulate flux through the neuronal endosomal pathway by decompressing potential "traffic jams." Exosome secretion may have the added benefit of unburdening the neuron's lysosomal system by delivering endosomal-lysosomal material into the extracellular space, where other cell types may contribute to the degradation of neuronal debris. Thus, maintaining robust neuronal exosome production may prevent or mitigate endosomal and lysosomal abnormalities linked to aging and neurodegenerative diseases. While the current evidence suggests that the exosomal system in the brain can be modulated both by membrane lipid composition and the expression of key proteins that contribute to the formation and secretion of exosomes, how exosomal pathway-regulatory elements sense and respond to perturbations in the endosomal pathway is not well understood. Based upon findings from the extensively studied DS and APOE4 models, we propose that enhanced neuronal exosome secretion can be a protective response, reducing pathological disruption of the endosomal-lysosomal system in disease-vulnerable neurons. Developing therapeutic approaches that help to maintain or enhance neuronal exosome biogenesis and release may be beneficial in a range of disorders of the central nervous system.

Apolipoprotein E (ApoE) is an important lipid carrier in both the periphery and the brain. The ApoE e4 allele (ApoE4) is the single most important genetic risk-factor for Alzheimer's disease (AD) while the e2 allele (ApoE2) is associated with a lower risk of AD-related neurodegeneration compared to the most common variant, e3 (ApoE3). ApoE genotype affects a variety of neural circuits; however, the olfactory system appears to provide early biomarkers of ApoE genotype effects. Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in 6-month- and 12-month-old mice with targeted replacement for the human ApoE2, ApoE3, or ApoE4 genes. Odor investigation and habituation were assessed, along with, olfactory bulb and piriform cortical local field potential activity. The results demonstrate that while initial odor investigation was unaffected by ApoE genotype, odor habituation was impaired in E4 relative to E2 mice, with E3 mice intermediate in function. There was also significant deterioration of odor habituation from 6 to 12 months of age regardless of the ApoE genotype. Olfactory system excitability and odor responsiveness were similarly determined by ApoE genotype, with an ApoE4 > ApoE3 > ApoE2 excitability ranking. The hyper-excitability of ApoE4 mice may contribute to the impairment of odor habituation memory, while the hypo-excitability of ApoE2 mice may contribute to its protective effects. Given that these ApoE mice do not have AD pathology, our results demonstrate the potential process by which ApoE affects the olfactory system at early stages, prior to the development of AD

Apolipoprotein E (ApoE) is an important lipid carrier in both the periphery and the brain. The ApoE ε4 allele (ApoE4) is the single most important genetic risk-factor for Alzheimer's disease (AD) while the ε 2 allele (ApoE2) is associated with a lower risk of AD-related neurodegeneration compared to the most common variant, ε 3 (ApoE3). ApoE genotype affects a variety of neural circuits; however, the olfactory system appears to provide early biomarkers of ApoE genotype effects. Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in 6-month- and 12-month-old mice with targeted replacement for the human ApoE2, ApoE3, or ApoE4 genes. Odor investigation and habituation were assessed, along with, olfactory bulb and piriform cortical local field potential activity. The results demonstrate that while initial odor investigation was unaffected by ApoE genotype, odor habituation was impaired in E4 relative to E2 mice, with E3 mice intermediate in function. There was also significant deterioration of odor habituation from 6 to 12 months of age regardless of the ApoE genotype. Olfactory system excitability and odor responsiveness were similarly determined by ApoE genotype, with an ApoE4 > ApoE3 > ApoE2 excitability ranking. Although motivated behavior is influenced by many processes, hyper-excitability of ApoE4 mice may contribute to impaired odor habituation, while hypo-excitability of ApoE2 mice may contribute to its protective effects. Given that these ApoE mice do not have AD pathology, our results demonstrate how ApoE affects the olfactory system at early stages, prior to the development of AD.

Extensive parenchymal and vascular Abeta deposits are pathological hallmarks of Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Abeta heterogeneity likely resulting from the action of multiple proteolytic enzymes. In spite of the numerous studies focusing in Abeta, the relevance of N- and C-terminal truncated species for AD pathogenesis remains largely understudied. In the present work, using novel antibodies specifically recognizing Abeta species N-terminally truncated at position 4 or C-terminally truncated at position 34, we provide a clear assessment of the differential topographic localization of these species in AD brains and transgenic models. Based on their distinct solubility, brain N- and C-terminal truncated species were extracted by differential fractionation and identified via immunoprecipitation coupled to mass spectrometry analysis. Biochemical/biophysical studies with synthetic homologues further confirmed the different solubility properties and contrasting fibrillogenic characteristics of the truncated species composing the brain Abeta peptidome. Abeta C-terminal degradation leads to the production of more soluble fragments likely to be more easily eliminated from the brain. On the contrary, N-terminal truncation at position 4 favors the formation of poorly soluble, aggregation prone peptides with high amyloidogenic propensity and the potential to exacerbate the fibrillar deposits, self-perpetuating the amyloidogenic loop. Detailed assessment of the molecular diversity of Abeta species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of Abeta truncations to the disease pathogenesis and their potential as novel therapeutic targets.

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

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

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.

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.

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

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.