Tau in the brain interstitial fluid is fragmented and seeding-competent.

In Alzheimer disease, Tau pathology is thought to propagate from cell to cell throughout interconnected brain areas. However, the forms of Tau released into the brain interstitial fluid (ISF) in vivo during the development of Tauopathy and their pathological relevance remain unclear. Combining in vivo microdialysis and biochemical analysis, we find that in Tau transgenic mice, human Tau (hTau) present in brain ISF is truncated and comprises at least 10 distinct fragments spanning the entire Tau protein. The fragmentation pattern is similar across different Tau transgenic models, pathological stages and brain areas. ISF hTau concentration decreases during Tauopathy progression, while its phosphorylation increases. ISF from mice with established Tauopathy induces Tau aggregation in HEK293-Tau biosensor cells. Notably, immunodepletion of ISF phosphorylated Tau, but not Tau fragments, significantly reduces its ability to seed Tau aggregation and only a fraction of Tau, separated by ultracentrifugation, is seeding-competent. These results indicate that ISF seeding competence is driven by a small subset of Tau, which potentially contribute to the propagation of Tau pathology.

Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1.

How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation­dependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.

Collecting antibodies and large molecule biomarkers in mouse interstitial brain fluid: a comparison of microdialysis and cerebral open flow microperfusion.

The determination of concentrations of large therapeutic molecules, like monoclonal antibodies (mAbs), in the interstitial brain fluid (ISF) is one of the cornerstones for the translation from preclinical species to humans of treatments for neurodegenerative diseases. Microdialysis (MD) and cerebral open flow microperfusion (cOFM) are the only currently available methods for extracting ISF, and their use and characterization for the collection of large molecules in rodents have barely started. For the first time, we compared both methods at a technical and performance level for measuring ISF concentrations of a non-target-binding mAb, trastuzumab, in awake and freely moving mice. Without correction of the data for recovery, concentrations of samples are over 10-fold higher through cOFM compared to MD. The overall similar pharmacokinetic profile and ISF exposure between MD (corrected for recovery) and cOFM indicate an underestimation of the absolute concentrations calculated with in vitro recovery. In vivo recovery (zero-flow rate method) revealed an increased extraction of trastuzumab at low flow rates and a 6-fold higher absolute concentration at steady state than initially calculated with the in vitro recovery. Technical optimizations have significantly increased the performance of both systems, resulting in the possibility of sampling up to 12 mice simultaneously. Moreover, strict aseptic conditions have played an important role in improving data quality. The standardization of these complex methods makes the unraveling of ISF concentrations attainable for various diseases and modalities, starting in this study with mAbs, but extending further in the future to RNA therapeutics, antibody-drug conjugates, and even cell therapies.

Cell-cell coupling and DNA methylation abnormal phenotypes in the after-hours mice.

BACKGROUND: DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. METHODS: In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. RESULTS: We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. CONCLUSIONS: The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

Phosphorylation of Parkin at serine 65 is essential for its activation in vivo.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand.

Dysregulated mitophagy has been linked to Parkinson's disease (PD) due to the role of PTEN-induced kinase 1 (PINK1) in mediating depolarization-induced mitophagy in vitro. Elegant mouse reporters have revealed the pervasive nature of basal mitophagy in vivo, yet the role of PINK1 and tissue metabolic context remains unknown. Using mito-QC, we investigated the contribution of PINK1 to mitophagy in metabolically active tissues. We observed a high degree of mitophagy in neural cells, including PD-relevant mesencephalic dopaminergic neurons and microglia. In all tissues apart from pancreatic islets, loss of Pink1 did not influence basal mitophagy, despite disrupting depolarization-induced Parkin activation. Our findings provide the first in vivo evidence that PINK1 is detectable at basal levels and that basal mammalian mitophagy occurs independently of PINK1. This suggests multiple, yet-to-be-discovered pathways orchestrating mammalian mitochondrial integrity in a context-dependent fashion, and this has profound implications for our molecular understanding of vertebrate mitophagy.

The Anthelmintic Drug Niclosamide and Its Analogues Activate the Parkinson's Disease Associated Protein Kinase PINK1.

Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early-onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases, such as PD. Herein, it is shown that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells through the reversible impairment of the mitochondrial membrane potential. With these compounds, for the first time, it is demonstrated that the PINK1 pathway is active and detectable in primary neurons. These findings suggest that niclosamide and its analogues are robust compounds for the study of the PINK1 pathway and may hold promise as a therapeutic strategy in PD and related disorders.

With mouse age comes wisdom: A review and suggestions of relevant mouse models for age-related conditions.

Ageing is a complex multifactorial process that results in many changes in physiological changes processes that ultimately increase susceptibility to a wide range of diseases. As such an ageing population is resulting in a pressing need for more and improved treatments across an assortment of diseases. Such treatments can come from a better understanding of the pathogenic pathways which, in turn, can be derived from models of disease. Therefore the more closely the model resembles the disease situation the more likely relevant the data will be that is generated from them. Here we review the state of knowledge of mouse models of a range of diseases and aspects of an ageing physiology that are all germane to ageing. We also give recommendations on the most common mouse models on their relevance to the clinical situations occurring in aged patients and look forward as to how research in ageing models can be carried out. As we continue to elucidate the pathophysiology of disease, often through mouse models, we also learn what is needed to refine these models. Such factors can include better models, reflecting the ageing patient population, or a better phenotypic understanding of existing models.

Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy.

BACKGROUND: Alzheimer disease (AD) and other tauopathies develop cerebral intracellular inclusions of hyperphosphorylated tau. Epidemiological and experimental evidence suggests a clear link between type 2 diabetes mellitus and AD. In AD animal models, tau pathology is exacerbated by metabolic comorbidities, such as insulin resistance and diabetes. Within this context, anitidiabetic drugs, including the widely-prescribed insulin-sensitizing drug metformin, are currently being investigated for AD therapy. However, their efficacy for tauopathy in vivo has not been tested. RESULTS: Here, we report that in the P301S mutant human tau (P301S) transgenic mouse model of tauopathy, chronic administration of metformin exerts paradoxical effects on tau pathology. Despite reducing tau phosphorylation in the cortex and hippocampus via AMPK/mTOR and PP2A, metformin increases insoluble tau species (including tau oligomers) and the number of inclusions with ß-sheet aggregates in the brain of P301S mice. In addition, metformin exacerbates hindlimb atrophy, increases P301S hyperactive behavior, induces tau cleavage by caspase 3 and disrupts synaptic structures. CONCLUSIONS: These findings indicate that metformin pro-aggregation effects mitigate the potential benefits arising from its dephosphorylating action, possibly leading to an overall increase of the risk of tauopathy in elderly diabetic patients.

Tau-Driven Neuronal and Neurotrophic Dysfunction in a Mouse Model of Early Tauopathy.

Tauopathies are neurodegenerative diseases characterized by intraneuronal inclusions of hyperphosphorylated tau protein and abnormal expression of brain-derived neurotrophic factor (BDNF), a key modulator of neuronal survival and function. The severity of both these pathological hallmarks correlate with the degree of cognitive impairment in patients. However, how tau pathology specifically modifies BDNF signaling and affects neuronal function during early prodromal stages of tauopathy remains unclear. Here, we report that the mild tauopathy developing in retinal ganglion cells (RGCs) of the P301S tau transgenic (P301S) mouse induces functional retinal changes by disrupting BDNF signaling via the TrkB receptor. In adult P301S mice, the physiological visual response of RGCs to pattern light stimuli and retinal acuity decline significantly. As a consequence, the activity-dependent secretion of BDNF in the vitreous is impaired in P301S mice. Further, in P301S retinas, TrkB receptors are selectively upregulated, but uncoupled from downstream extracellular signal-regulated kinase (ERK) 1/2 signaling. We also show that the impairment of TrkB signaling is triggered by tau pathology and mediates the tau-induced dysfunction of visual response. Overall our results identify a neurotrophin-mediated mechanism by which tau induces neuronal dysfunction during prodromal stages of tauopathy and define tau-driven pathophysiological changes of potential value to support early diagnosis and informed therapeutic decisions. SIGNIFICANCE STATEMENT: This work highlights the potential molecular mechanisms by which initial tauopathy induces neuronal dysfunction. Combining clinically used electrophysiological techniques (i.e., electroretinography) and molecular analyses, this work shows that in a relevant model of early tauopathy, the retina of the P301S mutant human tau transgenic mouse, mild tau pathology results in functional changes of neuronal activity, likely due to selective impairment of brain-derived neurotrophic factor signaling via its receptor, TrkB. These findings may have important translational implications for early diagnosis in a subset of Alzheimer's disease patients with early visual symptoms and emphasize the need to clarify the pathophysiological changes associated with distinct tauopathy stages to support informed therapeutic decisions and guide drug discovery.

CLIC1 functional expression is required for cAMP-induced neurite elongation in post-natal mouse retinal ganglion cells.

During neuronal differentiation, axonal elongation is regulated by both external and intrinsic stimuli, including neurotropic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability. Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance. Here, we investigated the expression of CLIC1 in primary hippocampal neurons and retinal ganglion cells (RGCs) and examined how the functional expression of CLIC1 specifically modulates neurite outgrowth of neonatal murine RGCs. Using a combination of electrophysiology and immunohistochemistry, we found that CLIC1 is expressed in hippocampal neurons and RGCs and that the chloride current mediated by CLIC1 is required for maintaining growth cone morphology and sustaining cAMP-stimulated neurite elongation in dissociated immunopurified RGCs. In cultured RGCs, inhibition of CLIC1 ionic current through the pharmacological blocker IAA94 or a specific anti-CLIC1 antibody directed against its extracellular domain prevents the neurite outgrowth induced by cAMP. CLIC1-mediated chloride current, which results from an increased open probability of the channel, is detected only when cAMP is elevated. Inhibition of protein kinase A prevents such current. These results indicate that CLIC1 functional expression is regulated by cAMP via protein kinase A and is required for neurite outgrowth modulation during neuronal differentiation. Using a combination of electrophysiology and immunohistochemistry, we found that the chloride intracellular channel 1 (CLIC1) protein modulates the speed of neurite growth. The chloride current mediated by CLIC1 is essential for maintaining growth cone morphology and is required for sustaining cAMP-stimulated neurite elongation in dissociated immunopurified neurons. The presence of either the CLIC1 current blocker IAA94 or the anti-CLIC1 antibody inhibits neurite growth of Retina Ganglion Cells cultured in the presence of 10 micromolar forskolin for 24 h.

Upregulation of presenilin 1 in brains of sporadic, late-onset Alzheimer's disease.

The activity of the ß-secretase involved in the cleavage of amyloid-ß (Aß) is increased in sporadic late-onset Alzheimer's disease (AD). Whether the corresponding γ-secretase activity is altered is still uncertain. We evaluated mRNA expression and protein levels of presenilin 1 (PS1) and γ-secretase activity in the frontal cortex of 32 cases with late-onset sporadic AD and those of 29 control subjects. We found a significant increase in PS1 mRNA, protein levels and γ-secretase activity in AD cases. These findings suggest that upregulation of PS1 leads to Aß overproduction and accumulation in sporadic AD.