Tau reduction with artificial microRNAs modulates neuronal physiology and improves tauopathy phenotypes in mice.

Abnormal tau accumulation is the hallmark of several neurodegenerative diseases, named tauopathies. Strategies aimed at reducing tau in the brain are promising therapeutic interventions, yet more precise therapies would require targeting specific nuclei and neuronal subpopulations affected by disease while avoiding global reduction of physiological tau. Here, we developed artificial microRNAs directed against the human MAPT mRNA to dwindle tau protein by engaging the endogenous RNA interference pathway. In human differentiated neurons in culture, microRNA-mediated tau reduction diminished neuronal firing without affecting neuronal morphology or impairing axonal transport. In the htau mouse model of tauopathy, we locally expressed artificial microRNAs in the prefrontal cortex (PFC), an area particularly vulnerable to initiating tau pathology in this model. Tau knockdown prevented the accumulation of insoluble and hyperphosphorylated tau, modulated firing activity of putative pyramidal neurons, and improved glucose uptake in the PFC. Moreover, such tau reduction prevented cognitive decline in aged htau mice. Our results suggest target engagement of designed tau-microRNAs to effectively reduce tau pathology, providing a proof of concept for a potential therapeutic approach based on local tau knockdown to rescue tauopathy-related phenotypes.

Mitochondrial vulnerability to oxidation in human brain organoids modelling Alzheimer's disease.

Reactive Oxygen Species (ROS) and mitochondrial dysfunction are implicated in the pathogenesis of Alzheimer's disease (AD), a common neurodegenerative disorder characterized by abnormal metabolism of the amyloid precursor protein (APP) in brain tissue. However, the exact mechanism by which abnormal APP leads to oxidative distress remains unclear. Damage to mitochondrial membrane and inhibition of mitochondrial respiration are thought to contribute to the progression of the disease. However, the lack of suitable human models that replicate pathological features, together with impaired cellular pathways, constitutes a major challenge in AD studies. In this work, we induced pluripotency in patient-derived skin fibroblasts carrying the Swedish mutation in App (APPswe), to generate human brain organoids that model AD, and studied redox regulation and mitochondrial homeostasis. We found time-dependent increases in AD-related pathological hallmarks in APPswe brain organoids, including elevated Aß levels, increased extracellular amyloid deposits, and enhanced tau phosphorylation. Interestingly, using live-imaging spinning-disk confocal microscopy, we found an increase in mitochondrial fragmentation and a significant loss of mitochondrial membrane potential in APPswe brain organoids when subjected to oxidative conditions. Moreover, ratiometric dyes in a live imaging setting revealed a selective increase in mitochondrial superoxide anion and hydrogen peroxide levels in APPswe brain organoids that were coupled to impairments in cytosolic and mitochondrial redoxin protein expression. Our results suggest a selective increase in mitochondrial vulnerability to oxidative conditions in APPswe organoids, indicating that the abnormal metabolism of APP leads to specific changes in mitochondrial homeostasis that enhance the vulnerability to oxidation in AD.

Modeling Alzheimer's Disease Using Human Brain Organoids.

Alzheimer's disease (AD) is the primary cause of dementia, to date. The urgent need to understand the biological and biochemical processes related to this condition, as well as the demand for reliable in vitro models for drug screening, has led to the development of novel techniques, among which stem cell methods are of utmost relevance for AD research, particularly the development of human brain organoids. Brain organoids are three-dimensional cellular aggregates derived from induced pluripotent stem cells (iPSCs) that recreate different neural cell interactions and tissue characteristics in culture. Here, we describe the protocol for the generation of brain organoids derived from AD patients and for the analysis of AD-derived pathology. AD organoids can recapitulate beta-amyloid and tau pathological features, making them a promising model for studying the molecular mechanisms underlying disease and for in vitro drug testing.

DYRK1A regulates the bidirectional axonal transport of APP in human-derived neurons.

DYRK1A triplication in Down's Syndrome (DS) and its overexpression in Alzheimer's Disease (AD) suggest a role for increased DYR1A activity in the abnormal metabolism of APP. Transport defects are early phenotypes in the progression of AD, which lead to APP processing impairments. However, whether DYRK1A regulates the intracellular transport and delivery of APP in human neurons remains unknown. From a proteomic dataset of human cerebral organoids treated with harmine, a DYRK1A inhibitor, we found expression changes in protein clusters associated with the control of microtubule-based transport and in close interaction with the APP vesicle. Live-imaging of APP axonal transport in human-derived neurons treated with harmine or overexpressing a dominant negative DYRK1A revealed a reduction in APP vesicle density and enhanced the stochastic behavior of retrograde vesicle transport. Moreover, harmine increased the fraction of slow segmental velocities and changed speed transitions supporting a DYRK1A-mediated effect in the exchange of active motor configuration. Contrarily, the overexpression of DYRK1A in human polarized neurons increased the axonal density of APP vesicles and enhanced the processivity of retrograde APP. In addition, increased DYRK1A activity induced faster retrograde segmental velocities together with significant changes in slow to fast anterograde and retrograde speeds transitions suggesting the facilitation of the active motor configuration. Our results highlight DYRK1A as a modulator of the axonal transport machinery driving APP intracellular distribution in neurons, and stress DYRK1A inhibition as a putative therapeutic intervention to restore APP axonal transport in DS and AD.Significance StatementAxonal transport defects are early events in the progression of neurodegenerative diseases such as Alzheimer's Disease (AD). However, the molecular mechanisms underlying transport defects remain elusive. DYRK1A kinase is triplicated in Down's Syndrome and overexpressed in AD, suggesting that DYRK1A dysfunction affects molecular pathways leading to early-onset neurodegeneration. Here, we show by live imaging of human-derived neurons that DYRK1A activity differentially regulates the intracellular trafficking of the amyloid precursor protein (APP). Further, single particle analysis revealed DYRK1A as a modulator of axonal transport and the configuration of active motors within the APP vesicle. Our work highlights DYRK1A as a regulator of APP axonal transport and metabolism; supporting DYRK1A inhibition as a therapeutic strategy to restore intracellular dynamics in AD.

Kinesin-1-mediated axonal transport of CB1 receptors is required for cannabinoid-dependent axonal growth and guidance.

Endocannabinoids (eCB) modulate growth cone dynamics and axonal pathfinding through the stimulation of cannabinoid type-1 receptors (CB1R), the function of which depends on their delivery and precise presentation at the growth cone surface. However, the mechanism involved in the axonal transport of CB1R and its transport role in eCB signaling remains elusive. As mutations in the kinesin-1 molecular motor have been identified in patients with abnormal cortical development and impaired white matter integrity, we studied the defects in axonal pathfinding and fasciculation in mice lacking the kinesin light chain 1 (Klc1-/-) subunit of kinesin-1. Reduced levels of CB1R were found in corticofugal projections and axonal growth cones in Klc1-/- mice. By live-cell imaging of CB1R-eGFP we characterized the axonal transport of CB1R vesicles and described the defects in transport that arise after KLC1 deletion. Cofilin activation, which is necessary for actin dynamics during growth cone remodeling, is impaired in the Klc1-/- cerebral cortex. In addition, Klc1-/- neurons showed expanded growth cones that were unresponsive to CB1R-induced axonal elongation. Together, our data reveal the relevance of kinesin-1 in CB1R axonal transport and in eCB signaling during brain wiring.

Proteasome stress leads to APP axonal transport defects by promoting its amyloidogenic processing in lysosomes.

Alzheimer disease (AD) pathology includes the accumulation of poly-ubiquitylated (also known as poly-ubiquitinated) proteins and failures in proteasome-dependent degradation. Whereas the distribution of proteasomes and its role in synaptic function have been studied, whether proteasome activity regulates the axonal transport and metabolism of the amyloid precursor protein (APP), remains elusive. By using live imaging in primary hippocampal neurons, we showed that proteasome inhibition rapidly and severely impairs the axonal transport of APP. Fluorescence cross-correlation analyses and membrane internalization blockage experiments showed that plasma membrane APP does not contribute to transport defects. Moreover, by western blotting and double-color APP imaging, we demonstrated that proteasome inhibition precludes APP axonal transport by enhancing its endo-lysosomal delivery, where ß-cleavage is induced. Taken together, we found that proteasomes control the distal transport of APP and can re-distribute Golgi-derived vesicles to the endo-lysosomal pathway. This crosstalk between proteasomes and lysosomes regulates the intracellular APP dynamics, and defects in proteasome activity can be considered a contributing factor that leads to abnormal APP metabolism in AD.This article has an associated First Person interview with the first author of the paper.