Cognitive phenotype and neurodegeneration associated with Tau in Huntington's disease.

OBJECTIVE: The clinical phenotype of Huntington's disease (HD) can be very heterogeneous between patients, even when they share equivalent CAG repeat length, age, or disease burden. This heterogeneity is especially evident in terms of the cognitive profile and related brain changes. To shed light on the mechanisms participating in this heterogeneity, the present study delves into the association between Tau pathology and more severe cognitive phenotypes and brain damage in HD. METHODS: We used a comprehensive neuropsychological examination to characterize the cognitive phenotype of a sample of 30 participants with early-to-middle HD for which we also obtained 3 T structural magnetic resonance image (MRI) and cerebrospinal fluid (CSF). We quantified CSF levels of neurofilament light chain (NfL), total Tau (tTau), and phosphorylated Tau-231 (pTau-231). Thanks to the cognitive characterization carried out, we subsequently explored the relationship between different levels of biomarkers, the cognitive phenotype, and brain integrity. RESULTS: The results confirmed that more severe forms of cognitive deterioration in HD extend beyond executive dysfunction and affect processes with clear posterior-cortical dependence. This phenotype was in turn associated with higher CSF levels of tTau and pTau-231 and to a more pronounced pattern of posterior-cortical atrophy in specific brain regions closely linked to the cognitive processes affected by Tau. INTERPRETATION: Our findings reinforce the association between Tau pathology, cognition, and neurodegeneration in HD, emphasizing the need to explore the role of Tau in the cognitive heterogeneity of the disease.

BACE2 beyond ß-processing of APP, its neuroprotective role in cerebrovascular endothelium.

Several proteases are involved in the proteolytic processing of the amyloid precursor protein (APP) generating the amyloidogenic Aß peptide, which can act as the triggering pathological effector of Alzheimer's disease (AD). Among these proteases, the ß-site amyloid precursor protein cleaving enzyme 2 (BACE2) is of particular interest because it was first proposed as an alternative ß-secretase to its homolog BACE1; however, accumulating evidence suggests that BACE2 acts as a non-amyloidogenic α-secretase and exerts neuroprotective effects. In this issue of J Neurochem, Katusic et al. present an interesting article reporting that BACE2 plays a role in preservation of cerebral vascular endothelial nitric oxide synthase (eNOS) function, thus exerting protective functions. Their data support that the process is mediated by the large soluble non-amyloidogenic APP fragment sAPPα through the γ-aminobutyric acid type B receptor 1, which enhances the expression of a major transcription factor for eNOS gene expression in endothelial cells, the Krüppel-like factor 2. These protective functions of BACE2 contrast with the pathogenic role of BACE1 as a key player in the AD amyloidogenic pathway. Indeed, many efforts have been invested in BACE1 inhibitors as potential disease modifiers for AD. Unfortunately, the results in clinical trials have been disappointing. In this scenario, a better understanding of the functions of BACE2, as well as the selectivity of BACE1 inhibitors with respect to other ß-secretases (mainly BACE2), is crucial for the development of new therapeutic agents. Furthermore, specific cellular targeting should also be considered to improve such therapies due to the diverse balance of secretases targeting APP and the complex cross-talk between them and the generated APP fragments.

Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma.

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.

Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues.

Extracellular vesicles (EVs) are nanoscale vesicles secreted into the extracellular space by all cell types, including neurons and astrocytes in the brain. EVs play pivotal roles in physiological and pathophysiological processes such as waste removal, cell-to-cell communication and transport of either protective or pathogenic material into the extracellular space. Here we describe a detailed protocol for the reliable and consistent isolation of EVs from both murine and human brains, intended for anyone with basic laboratory experience and performed in a total time of 27 h. The method includes a mild extracellular matrix digestion of the brain tissue, a series of filtration and centrifugation steps to purify EVs and an iodixanol-based high-resolution density step gradient that fractionates different EV populations, including mitovesicles, a newly identified type of EV of mitochondrial origin. We also report detailed downstream protocols for the characterization and analysis of brain EV preparations using nanotrack analysis, electron microscopy and western blotting, as well as for measuring mitovesicular ATP kinetics. Furthermore, we compared this novel iodixanol-based high-resolution density step gradient to the previously described sucrose-based gradient. Although the yield of total EVs recovered was similar, the iodixanol-based gradient better separated distinct EV species as compared with the sucrose-based gradient, including subpopulations of microvesicles, exosomes and mitovesicles. This technique allows quantitative, highly reproducible analyses of brain EV subtypes under normal physiological processes and pathological brain conditions, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

Sex Differentially Alters Secretion of Brain Extracellular Vesicles During Aging: A Potential Mechanism for Maintaining Brain Homeostasis.

Extracellular vesicles (EVs) in the brain play a role in neuronal homeostasis by removing intracellular material and regulating cell-to-cell communication. Given that sex and aging differentially modulate brain networks, we investigated sex-dependent differences in EV levels and content in the brain during aging. EVs were isolated from the brains of 3, 6, 12, 18, and 24 month-old female and male C57BL/6 J mice, and the levels of different EV species determined. While the number of plasma membrane-derived microvesicles and a subset of late endosomes-derived exosomes increased with age in the brain of female mice, no significant changes were seen in males. Mitochondria-derived mitovesicles in the brain increased during aging in both sexes, a change that may reflect aging-dependent alterations in mitochondrial function. These findings reveal enhanced turnover during aging in female brains, suggesting a mechanism for advantageous successful female brain aging and sex-depending different susceptibility to age-related neurodegenerative diseases.

Dissociable contribution of plasma NfL and p-tau181 to cognitive impairment in Parkinson's disease.

BACKGROUND: Cognitive dysfunction is a disabling complication in Parkinson's disease (PD). Accuracy of diagnosis of mild cognitive impairment in PD (PD-MCI) depends on the tests performed, which limits results generalization. Blood-based biomarkers could provide additional objective information for PD-MCI diagnosis and progression. Blood neurofilament light chain (NfL), a marker of neuronal injury, has shown good performance for PD disease stratification and progression. While NfL is not disease-specific, phosphorylated-tau at threonine-181 (p-tau181) in blood is a highly specific marker of concomitant brain amyloid-ß and tau pathology. METHODS: We investigated the potential of plasma NfL and p-tau181 levels as markers of cognitive impairment in a prospective cohort of 109 PD patients with and without PD-MCI (age 68.1 ± 7 years, education 12.2± 5 years), and 40 comparable healthy controls. After a follow-up of 4 years, we evaluated their predictive value for progression to dementia. RESULTS: Although NfL and p-tau181 levels were significantly increased in PD compared with healthy controls, only NfL levels were significantly higher in PD-MCI compared with PD with normal cognition (PD-NC) at baseline. After a follow-up of 4 years, only NfL predicted progression to dementia (HR 1.23, 95% CI 1.02-1.53; p = 0.038). Significant correlations between fluid biomarkers and neuropsychological examination were only found with NfL levels. CONCLUSIONS: Plasma NfL levels objectively differentiates PD-MCI from PD-NC patients, and may serve as a plasma biomarker for predicting progression to dementia in PD. Plasma levels of p-tau181 does not seem to help in differentiating PD-MCI or to predict future cognitive deterioration.

Plasma TDP-43 Reflects Cortical Neurodegeneration and Correlates with Neuropsychiatric Symptoms in Huntington's Disease.

PURPOSE: Huntington's disease (HD) is a monogenic neurodegenerative disease with no effective treatment currently available. The pathological hallmark of HD is the aggregation of mutant huntingtin in the medium spiny neurons of the striatum, leading to severe subcortical atrophy. Cortical degeneration also occurs in HD from its very early stages, although its biological origin is poorly understood. Among the possible pathological mechanisms that could promote cortical damage in HD, the in vivo study of TDP-43 pathology remains to be explored, which was the main objective of this work. METHODS: We investigated the clinical and structural brain correlates of plasma TDP-43 levels in a sample of 36 HD patients. Neuroimaging alterations were assessed both at the macrostructural (cortical thickness) and microstructural (intracortical diffusivity) levels. Importantly, we controlled for mutant huntingtin and tau biomarkers in order to assess the independent role of TDP-43 in HD neurodegeneration. RESULTS: Plasma TDP-43 levels in HD specifically correlated with the presence and severity of apathy (p = 0.003). The TDP-43 levels also reflected cortical thinning and microstructural degeneration, especially in frontal and anterior-temporal regions (p < 0.05 corrected). These TDP-43-related brain alterations correlated, in turn, with the severity of cognitive, motor and behavioral symptoms. CONCLUSION: Our results suggest that the presence of TDP-43 pathology in HD has an independent contribution to the severity of neuropsychiatric symptoms and frontotemporal degeneration. These findings point out the importance of TDP-43 as an additional pathological process to be taken into consideration in this devastating disorder.

Interaction between sex and neurofilament light chain on brain structure and clinical severity in Huntington's disease.

Female Huntington's disease (HD) patients have consistently shown a faster clinical worsening than male, but the underlying mechanisms responsible for this observation remain unknown. Here, we describe how sex modifies the impact of neurodegeneration on brain atrophy and clinical severity in HD. Cerebrospinal fluid neurofilament light chain (NfL) levels were used as a biological measure of neurodegeneration, and brain atrophy was assessed by structural magnetic resonance imaging. We found that larger NfL values in women reflect higher brain atrophy and clinical severity than in men (p < 0.05 for an interaction model). This differential vulnerability could have important implications in clinical trials.

Cortical microstructural correlates of plasma neurofilament light chain in Huntington's disease.

INTRODUCTION: Huntington's disease (HD) is a severe neurodegenerative disorder with no effective treatment. Minimally-invasive biomarkers such as blood neurofilament light chain (NfL) in HD are therefore needed to quantitatively characterize neuronal loss. NfL levels in HD are known to correlate with disease progression and striatal atrophy, but whether they also reflect cortical degeneration remains elusive. METHODS: In a sample of 35 HD patients, we characterized the cortical macro (cortical thickness) and microstructural (increased intracortical diffusivity) correlates of plasma NfL levels. We further investigated whether NfL-related cortical alterations correlated with clinical indicators of disease progression. RESULTS: Increased plasma NfL levels in HD reflected posterior-cortical microstructural degeneration, but not reduced cortical thickness (p < 0.05, corrected). Importantly, these imaging alterations correlated, in turn, with more severe motor, cognitive and behavioral symptoms. CONCLUSION: Plasma NfL levels may be useful for tracking clinically-meaningful cortical deterioration in HD. Additionally, our results further reinforce the role of intracortical diffusivity as a valuable imaging indicator in movement disorders.

Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome.

Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer's disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs "mitovesicles." We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.

Extracellular vesicles: where the amyloid precursor protein carboxyl-terminal fragments accumulate and amyloid-ß oligomerizes.

Pleiotropic roles are proposed for brain extracellular vesicles (EVs) in the development of Alzheimer's disease (AD). Our previous studies have suggested a beneficial role for EVs in AD, where the endosomal system in vulnerable neurons is compromised, contributing to the removal of accumulated material from neurons. However, the involvement of EVs in propagating AD amyloidosis throughout the brain has been considered because the amyloid-ß precursor protein (APP), APP metabolites, and key APP cleaving enzymes were identified in association with EVs. Here, we undertook to determine whether the secretase machinery is actively processing APP in EVs isolated from the brains of wild-type and APP overexpressing Tg2576 mice. We found that full-length APP is cleaved in EVs incubated in the absence of cells. The resulting metabolites, both α- and ß-APP carboxyl-terminal fragments and APP intracellular domain accumulate in EVs over time and amyloid-ß dimerizes. Thus, EVs contribute to the removal from neurons and transport of APP-derived neurotoxic peptides. While this is potentially a venue for propagation of the pathology throughout the brain, it may contribute to efficient removal of neurotoxic peptides from the brain.

Increased plasma neurofilament light chain levels in patients with type-1 diabetes with impaired awareness of hypoglycemia.

INTRODUCTION: Impaired awareness of hypoglycemia (IAH) is a common complication in patients with type-1 diabetes (T1D). IAH is a major risk factor for severe hypoglycemic events, leading to adverse clinical consequences and cerebral damage. Non-invasive, cost-effective, and logistically efficient biomarkers for this condition have not been validated. Here, we propose plasma neurofilament light chain (NfL) levels as a biomarker of neuroaxonal damage in patients with T1D-IAH. RESEARCH DESIGN AND METHODS: 54 patients were included into the study (18 T1D-IAH, 18 T1D with normal awareness of hypoglycemia (NAH) and 18 healthy controls). We measured plasma NfL levels and studied cerebral gray matter alterations on MRI. RESULTS: We found that NfL levels were increased in patients with T1D-IAH compared with patients with T1D-NAH and healthy controls. Importantly, increased NfL levels correlated with reduced cerebral gray matter volume and increased IAH severity in patients with T1D-IAH. CONCLUSION: Overall, our findings identify plasma NfL levels as a potential biomarker of cerebral damage in this population, motivating further confirmatory studies with potential implications in clinical trials.

Serum neurofilament light chain levels reflect cortical neurodegeneration in de novo Parkinson's disease.

INTRODUCTION: Cognitive impairment and dementia are highly prevalent non-motor complications in Parkinson's disease (PD) with deleterious consequences for patients and caregivers. With no treatment currently available, finding and validating minimally-invasive biomarkers of neurodegeneration in this population represents an urgent need for clinical trials targeting its prevention or delay. Recently, serum neurofilament light chain (NfL) levels have been identified as a promising biomarker of neural loss, but whether they reflect cortical neurodegeneration in early PD stages has not been addressed. METHODS: From the Parkinson's Progression Markers Initiative (PPMI), we selected 133 de novo PD patients and 56 healthy controls (HC) with available structural neuroimaging and serum NfL data. We then studied whether NfL levels were abnormal in the PD group with respect to HC, and whether they correlated with cognitive indicators and cortical macro (cortical thinning) and microstructural (increased intracortical mean diffusivity) degeneration. RESULTS: Serum NfL levels were significantly increased in the PD group (p = 0.010), and were also related to worse cognitive performance and a cortical macro and microstructural compromise (p < 0.05 corrected). These associations were observed both cross-sectionally and longitudinally within a one-year follow-up period. Topographically, NfL levels reflected posterior-cortical deterioration rather than frontal damage. Importantly, NfL levels were not associated with striatal SPECT-DAT uptake or ß-amyloid burden. DISCUSSION: Our results show that serum NfL levels reflect cortical neurodegeneration from the very early stages of PD. Moreover, its brain structural correlates and its lack of relationship with dopaminergic depletion or amyloidosis suggests that NfL could track the underlying pathological process leading to PD dementia.

CLU rs11136000 promotes early cognitive decline in Parkinson's disease.

BACKGROUND: The C allele of the rs11136000 genetic variant of the clusterin gene has been associated with increased risk of Alzheimer's disease. However, a comprehensive characterization of the role of this genetic variant in early cognitive deterioration in PD is lacking. METHODS: Using the Parkinson's Progression Markers Initiative database, we compared baseline and 5-year cognitive performance between high-risk and low-risk clusterin genotypes. RESULTS: At baseline, recently diagnosed and drug-naive de novo PD patients with the high-risk clusterin genotype showed lower cognitive scores in memory and executive function tests. These differences were even higher at the 5-year follow-up, when they showed a higher prevalence of clinically diagnosed mild cognitive impairment or dementia. They also showed cortical thinning at baseline and increased annual thinning in frontal and posterior cortical regions. DISCUSSION: Our results provide evidence of this clusterin genotype promoting early cognitive deterioration in PD, but further research is needed to delineate the specific neurodegenerative pathways underlying this clinical association. © 2020 International Parkinson and Movement Disorder Society.

A pleiotropic role for exosomes loaded with the amyloid ß precursor protein carboxyl-terminal fragments in the brain of Down syndrome patients.

Down syndrome (DS) is characterized by cognitive deficits throughout the life span and with the development of aging-dependent Alzheimer's type neuropathology, which is related to the triplication of the amyloid ß precursor protein (APP) gene. A dysfunctional endosomal system in neurons is an early characteristic of DS and APP metabolites accumulate in endosomes in DS neurons. We have previously shown enhanced release of exosomes in the brain of DS patients and the mouse model of DS Ts[Rb(12.1716)]2Cje (Ts2), and by DS fibroblasts, as compared with diploid controls. Here, we demonstrate that exosome-enriched extracellular vesicles (hereafter called EVs) isolated from DS and Ts2 brains, and from the culture media of human DS fibroblasts are enriched in APP carboxyl-terminal fragments (APP-CTFs) as compared with diploid controls. Moreover, APP-CTFs levels increase in an age-dependent manner in EVs isolated from the brain of Ts2 mice. The release of APP-CTFs-enriched exosomes may have a pathogenic role by transporting APP-CTFs into naïve neurons and propagating these neurotoxic metabolites, which are also a source of amyloid ß, throughout the brain, but also provides a benefit to DS neurons by shedding APP-CTFs accumulated intracellularly.

Neuroprotection mediated by cystatin C-loaded extracellular vesicles.

Cystatin C (CysC) is implicated in neuroprotection and repair in the nervous system in response to diverse neurotoxic conditions. In addition to being secreted from cells in a soluble form, CysC is released by cells in association with extracellular vesicles (EVs), including exosomes. We demonstrate that EVs containing CysC protect cultured cells from starvation-induced death. Moreover, while EVs secreted by CysC-deficient cells were not protective, EVs secreted by CysC-deficient cells treated with exogenous human CysC significantly enhanced the survival of the cells. CysC also plays a role in modulating the secretion of EVs, enhancing secretion of EVs by primary cortical neurons and primary cortical smooth muscle cells. Confirming these in vitro findings, higher EV levels were observed in the brain extracellular space of transgenic mice expressing human CysC as compared to littermate controls. Regulation of cell-secreted EV levels and content in the brain is likely to be essential to maintaining normal brain function. We propose that enhanced EV release could rescue the deleterious effects of dysfunction of the endosomal-lysosomal system in neurodegenerative disorders. Moreover, a higher level of CysC-loaded EVs released from cells in the central nervous system has important protective functions, representing a potential therapeutic tool for disorders of the central nervous system.

Enhanced generation of intraluminal vesicles in neuronal late endosomes in the brain of a Down syndrome mouse model with endosomal dysfunction.

Down syndrome (DS) is a human genetic disease caused by trisomy of chromosome 21 and characterized by early developmental brain abnormalities. Dysfunctional endosomal pathway in neurons is an early event of DS and Alzheimer's disease. Recently, we have demonstrated that exosome secretion is upregulated in human DS postmortem brains, in the brain of the trisomic mouse model Ts[Rb(12.1716 )]2Cje (Ts2) and by DS fibroblasts as compared with disomic controls. High levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Partially blocking exosome secretion by DS fibroblasts exacerbated a pre-existing early endosomal pathology. We thus hypothesized that enhanced CD63 expression induces generation of intraluminal vesicles (ILVs) in late endosomes/multivesicular bodies (MVBs), increasing exosome release as an endogenous mechanism to mitigate endosomal abnormalities in DS. Herein, we show a high-resolution electron microscopy analysis of MVBs in neurons of the frontal cortex of 12-month-old Ts2 mice and littermate diploid controls. Our quantitative analysis revealed that Ts2 MVBs are larger, more abundant, and contain a higher number of ILVs per neuron compared to controls. These findings were further corroborated biochemically by Western blot analysis of purified endosomal fractions showing higher levels of ILVs proteins in the same fractions containing endosomal markers in the brain of Ts2 mice compared to controls. These data suggest that upregulation of ILVs production may be a key homeostatic mechanism to alleviate endosomal dysregulation via the endosomal-exosomal pathway.

Intracellular metalloprotease activity controls intraneuronal Aß aggregation and limits secretion of Aß via exosomes.

Accumulating evidence suggests that the abnormal aggregation of amyloid-ß (Αß) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where Aß is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced Aß. In this study, we determined whether disruption of Aß catabolism could trigger Aß aggregation within neurons and increase the amount of Aß associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular Aß levels and promoted the intracellular formation of Aß oligomers, a process that did not require internalization of secreted Aß. In vivo, the accumulation of intraneuronal Aß aggregates was accompanied by increased levels of both extracellular and exosome-associated Aß, including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of Aß degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal Aß aggregates and their subsequent release into the extracellular space via exosomes.-Pacheco-Quinto, J., Clausen, D., Pérez-González, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal Aß aggregation and limits secretion of Aß via exosomes.

Apolipoprotein E4 genotype compromises brain exosome production.

In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the ɛ4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-ß and tau pathology. In human post-mortem tissue and mouse models humanized for apolipoprotein E, we examined the impact of apolipoprotein E4 expression on brain exosomes, vesicles that are produced within and secreted from late-endocytic multivesicular bodies. Compared to humans or mice homozygous for the risk-neutral ɛ3 allele we show that the ɛ4 allele, whether homozygous or heterozygous with an ɛ3 allele, drives lower exosome levels in the brain extracellular space. In mice, we show that the apolipoprotein E4-driven change in brain exosome levels is age-dependent: while not present at age 6 months, it is detectable at 12 months of age. Expression levels of the exosome pathway regulators tumor susceptibility gene 101 (TSG101) and Ras-related protein Rab35 (RAB35) were found to be reduced in the brain at the protein and mRNA levels, arguing that apolipoprotein E4 genotype leads to a downregulation of exosome biosynthesis and release. Compromised exosome production is likely to have adverse effects, including diminishing a cell's ability to eliminate materials from the endosomal-lysosomal system. This reduction in brain exosome levels in 12-month-old apolipoprotein E4 mice occurs earlier than our previously reported brain endosomal pathway changes, arguing that an apolipoprotein E4-driven failure in exosome production plays a primary role in endosomal and lysosomal deficits that occur in apolipoprotein E4 mouse and human brains. Disruption of these interdependent endosomal-exosomal-lysosomal systems in apolipoprotein E4-expressing individuals may contribute to amyloidogenic amyloid-ß precursor protein processing, compromise trophic signalling and synaptic function, and interfere with a neuron's ability to degrade material, all of which are events that lead to neuronal vulnerability and higher risk of Alzheimer's disease development. Together, these data suggest that exosome pathway dysfunction is a previously unappreciated component of the brain pathologies that occur as a result of apolipoprotein E4 expression.

Cystatin C prevents neuronal loss and behavioral deficits via the endosomal pathway in a mouse model of down syndrome.

Cystatin C (CysC) plays diverse protective roles under conditions of neuronal challenge. We investigated whether CysC protects from trisomy-induced pathologies in a mouse model of Down syndrome (DS), the most common cause of developmental cognitive and behavioral impairments in humans. We have previously shown that the segmental trisomy mouse model, Ts[Rb(12.1716)]2Cje (Ts2) has DS-like neuronal and behavioral deficiencies. The current study reveals that transgene-mediated low levels of human CysC overexpression has a preventive effect on numerous neuropathologies in the brains of Ts2 mice, including reducing early and late endosome enlargement in cortical neurons and decreasing loss of basal forebrain cholinergic neurons (BFCNs). Consistent with these cellular benefits, behavioral dysfunctions were also prevented, including deficits in nesting behavior and spatial memory. We determined that the CysC-induced neuroprotective mechanism involves activation of the phosphotidylinositol kinase (PI3K)/AKT pathway. Activating this pathway leads to enhanced clearance of accumulated endosomal substrates, protecting cells from DS-mediated dysfunctions in the endosomal system and, for BFCNs, from neurodegeneration. Our findings suggest that modulation of the PI3/AKT pathway offers novel therapeutic interventions for patients with DS.