Pathophysiologic abnormalities in transgenic mice carrying the Alzheimer disease PSEN1 Δ440 mutation.

Mutations in PSEN1 were first discovered as a cause of Alzheimer's disease (AD) in 1995, yet the mechanism(s) by which the mutations cause disease still remains unknown. The generation of novel mouse models assessing the effects of different mutations could aid in this endeavor. Here we report on transgenic mouse lines made with the Δ440 PSEN1 mutation that causes AD with parkinsonism:- two expressing the un-tagged human protein and two expressing a HA-tagged version.

mTOR inhibition in Q175 Huntington's disease model mice facilitates neuronal autophagy and mutant huntingtin clearance.

Huntington's disease (HD) is caused by expansion of the polyglutamine stretch in huntingtin protein (HTT) resulting in hallmark aggresomes/inclusion bodies (IBs) composed of mutant huntingtin protein (mHTT) and its fragments. Stimulating autophagy to enhance mHTT clearance is considered a potential therapeutic strategy for HD. Our recent evaluation of the autophagic-lysosomal pathway (ALP) in human HD brain reveals upregulated lysosomal biogenesis and relatively normal autophagy flux in early Vonsattel grade brains, but impaired autolysosome clearance in late grade brains, suggesting that autophagy stimulation could have therapeutic benefits as an earlier clinical intervention.

Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment.

Mitovesicles secreted into the extracellular space of brains with mitochondrial dysfunction impair synaptic plasticity.

Background: Hypometabolism tied to mitochondrial dysfunction occurs in the aging brain and in neurodegenerative disorders, including in Alzheimer's disease, in Down syndrome, and in mouse models of these conditions. We have previously shown that mitovesicles, small extracellular vesicles (EVs) of mitochondrial origin, are altered in content and abundance in multiple brain conditions characterized by mitochondrial dysfunction. However, given their recent discovery, it is yet to be explored what mitovesicles regulate and modify, both under physiological conditions and in the diseased brain.

Cocaine perturbs mitovesicle biology in the brain.

Cocaine, an addictive psychostimulant, has a broad mechanism of action, including the induction of a wide range of alterations in brain metabolism and mitochondrial homeostasis. Our group recently identified a subpopulation of non-microvesicular, non-exosomal extracellular vesicles of mitochondrial origin (mitovesicles) and developed a method to isolate mitovesicles from brain parenchyma. We hypothesised that the generation and secretion of mitovesicles is affected by mitochondrial abnormalities induced by chronic cocaine exposure.

Autophagy is a novel pathway for neurofilament protein degradation .

How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover .

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.

Reduced calcium levels and accumulation of abnormal insulin granules in stem cell models of HNF1A deficiency.

Mutations in HNF1A cause Maturity Onset Diabetes of the Young (HNF1A-MODY). To understand mechanisms of β-cell dysfunction, we generated stem cell-derived pancreatic endocrine cells with hypomorphic mutations in HNF1A. HNF1A-deficient β-cells display impaired basal and glucose stimulated-insulin secretion, reduced intracellular calcium levels in association with a reduction in CACNA1A expression, and accumulation of abnormal insulin granules in association with SYT13 down-regulation.

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.

Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques.

Autophagy is markedly impaired in Alzheimer's disease (AD). Here we reveal unique autophagy dysregulation within neurons in five AD mouse models in vivo and identify its basis using a neuron-specific transgenic mRFP-eGFP-LC3 probe of autophagy and pH, multiplex confocal imaging and correlative light electron microscopy. Autolysosome acidification declines in neurons well before extracellular amyloid deposition, associated with markedly lowered vATPase activity and build-up of Aβ/APP-βCTF selectively within enlarged de-acidified autolysosomes.

Cocaine Modulates the Neuronal Endosomal System and Extracellular Vesicles in a Sex-Dependent Manner.

In multiple neurodevelopmental and neurodegenerative disorders, endosomal changes correlate with changes in exosomes. We examined this linkage in the brain of mice that received cocaine injections for two weeks starting at 2.5 months of age.

Axonal transport of late endosomes and amphisomes is selectively modulated by local Ca efflux and disrupted by PSEN1 loss of function.

Dysfunction and mistrafficking of organelles in autophagy- and endosomal-lysosomal pathways are implicated in neurodegenerative diseases. Here, we reveal selective vulnerability of maturing degradative organelles (late endosomes/amphisomes) to disease-relevant local calcium dysregulation. These organelles undergo exclusive retrograde transport in axons, with occasional pauses triggered by regulated calcium efflux from agonist-evoked transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) channels-an effect greatly exaggerated by exogenous agonist mucolipin synthetic agonist 1 (ML-SA1).

Wolframin is a novel regulator of tau pathology and neurodegeneration.

Selective neuronal vulnerability to protein aggregation is found in many neurodegenerative diseases including Alzheimer's disease (AD). Understanding the molecular origins of this selective vulnerability is, therefore, of fundamental importance. Tau protein aggregates have been found in Wolframin (WFS1)-expressing excitatory neurons in the entorhinal cortex, one of the earliest affected regions in AD.

Numb is required for optimal contraction of skeletal muscle.

Background: The role of Numb, a protein that is important for cell fate and development and that, in human muscle, is expressed at reduced levels with advanced age, was investigated; adult mice skeletal muscle and its localization and function within myofibres were determined.

Methods: Numb expression was evaluated by western blot. Numb localization was determined by confocal microscopy.

Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons.

Lysosomal trafficking and maturation in neurons remain poorly understood and are unstudied in vivo despite high disease relevance. We generated neuron-specific transgenic mice to track vesicular CTSD acquisition, acidification, and traffic within the autophagic-lysosomal pathway in vivo, revealing that mature lysosomes are restricted from axons. Moreover, TGN-derived transport carriers (TCs), not lysosomes, supply lysosomal components to axonal organelles.

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.

Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease.

Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer's disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-β secretase cleaved C-terminal fragment (APP-βCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-βCTF in the PA-Rab5 mouse model. We report that Rab5 overactivation alone recapitulates diverse prodromal and degenerative features of AD.

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.

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-β 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.

An absence of nuclear lamins in keratinocytes leads to ichthyosis, defective epidermal barrier function, and intrusion of nuclear membranes and endoplasmic reticulum into the nuclear chromatin.

B-type lamins (lamins B1 and B2) have been considered to be essential for many crucial functions in the cell nucleus (e.g., DNA replication and mitotic spindle formation).

High-resolution imaging of dietary lipids in cells and tissues by NanoSIMS analysis.

Nanoscale secondary ion MS (NanoSIMS) imaging makes it possible to visualize stable isotope-labeled lipids in cells and tissues at 50 nm lateral resolution. Here we report the use of NanoSIMS imaging to visualize lipids in mouse cells and tissues. After administering stable isotope-labeled fatty acids to mice by gavage, NanoSIMS imaging allowed us to visualize neutral lipids in cytosolic lipid droplets in intestinal enterocytes, chylomicrons at the basolateral surface of enterocytes, and lipid droplets in cardiomyocytes and adipocytes.