MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons

Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK

The blood-brain barrier is dysregulated in COVID-19 and serves as a CNS entry route for SARS-CoV-2.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling

Loss of Homeostatic Microglia Signature in Prion Diseases

Prion diseases are neurodegenerative diseases that affect humans and animals. They are always fatal and, to date, no treatment exists. The hallmark of prion disease pathophysiology is the misfolding of an endogenous protein, the cellular prion protein (PrPC), into its disease-associated isoform PrPSc. Besides the aggregation and deposition of misfolded PrPSc, prion diseases are characterized by spongiform lesions and the activation of astrocytes and microglia. Microglia are the innate immune cells of the brain. Activated microglia and astrocytes represent a common pathological feature in neurodegenerative disorders. The role of activated microglia has already been studied in prion disease mouse models; however, it is still not fully clear how they contribute to disease progression. Moreover, the role of microglia in human prion diseases has not been thoroughly investigated thus far, and specific molecular pathways are still undetermined. Here, we review the current knowledge on the different roles of microglia in prion pathophysiology. We discuss microglia markers that are also dysregulated in other neurodegenerative diseases including microglia homeostasis markers. Data on murine and human brain tissues show that microglia are highly dysregulated in prion diseases. We highlight here that the loss of homeostatic markers may especially stand out

Neurobeachin and the Kinesin KIF21B Are Critical for Endocytic Recycling of NMDA Receptors and Regulate Social Behavior

Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology

Muskelin regulates actin-dependent synaptic changes and intrinsic brain activity relevant to behavioral and cognitive processes

Muskelin (Mkln1) is implicated in neuronal function, regulating plasma membrane receptor trafficking. However, its influence on intrinsic brain activity and corresponding behavioral processes remains unclear. Here we show that murine Mkln1 knockout causes non-habituating locomotor activity, increased exploratory drive, and decreased locomotor response to amphetamine. Muskelin deficiency impairs social novelty detection while promoting the retention of spatial reference memory and fear extinction recall. This is strongly mirrored in either weaker or stronger resting-state functional connectivity between critical circuits mediating locomotor exploration and cognition. We show that Mkln1 deletion alters dendrite branching and spine structure, coinciding with enhanced AMPAR-mediated synaptic transmission but selective impairment in synaptic potentiation maintenance. We identify muskelin at excitatory synapses and highlight its role in regulating dendritic spine actin stability. Our findings point to aberrant spine actin modulation and changes in glutamatergic synaptic function as critical mechanisms that contribute to the neurobehavioral phenotype arising from Mkln1 ablation

TRIM3 Regulates the Motility of the Kinesin Motor Protein KIF21B

<div><p>Kinesin superfamily proteins (KIFs) are molecular motors that transport cellular cargo along the microtubule cytoskeleton. KIF21B is a neuronal kinesin that is highly enriched in dendrites. The regulation and specificity of microtubule transport involves the binding of motors to individual cargo adapters and accessory proteins. Moreover, posttranslational modifications of either the motor protein, their cargos or tubulin regulate motility, cargo recognition and the binding or unloading of cargos. Here we show that the ubiquitin E3 ligase TRIM3, also known as BERP, interacts with KIF21B via its RBCC domain. TRIM3 is found at intracellular and Golgi-derived vesicles and co-localizes with the KIF21B motor in neurons. <i>Trim3</i> gene deletion in mice and TRIM3 overexpression in cultured neurons both suggested that the E3-ligase function of TRIM3 is not involved in KIF21B degradation, however TRIM3 depletion reduces the motility of the motor. Together, our data suggest that TRIM3 is a regulator in the modulation of KIF21B motor function.</p> </div

TRPM4 cation channel mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, an inflammatory disease of the central nervous system (CNS), axonal and neuronal loss are major causes for irreversible neurological disability. However, which molecules contribute to axonal and neuronal injury under inflammatory conditions remains largely unknown. Here we show that the transient receptor potential melastatin 4 (TRPM4) cation channel is crucial in this process. TRPM4 is expressed in mouse and human neuronal somata, but it is also expressed in axons in inflammatory CNS lesions in experimental autoimmune encephalomyelitis (EAE) in mice and in human multiple sclerosis tissue. Deficiency or pharmacological inhibition of TRPM4 using the antidiabetic drug glibenclamide resulted in reduced axonal and neuronal degeneration and attenuated clinical disease scores in EAE, but this occurred without altering EAE-relevant immune function. Furthermore, Trpm4(-/-) mouse neurons were protected against inflammatory effector mechanisms such as excitotoxic stress and energy deficiency in vitro. Electrophysiological recordings revealed TRPM4-dependent neuronal ion influx and oncotic cell swelling upon excitotoxic stimulation. Therefore, interference with TRPM4 could translate into a new neuroprotective treatment strategy

The E3 ligase TRIM3 is not involved in the degradation of the motor protein KIF21B.

<p>(A, B) KIF21B half life analysis using cycloheximide (CHX) chase experiments. More than 50% of KIF21B degrades within 48 hours in cultured hippocampal neurons (DIV16) derived from wildtype (+/+) mice. TRIM3 genetic depletion does not alter the half life of KIF21B, as assessed through evaluation of relative KIF21B signal intensities. Optineurin and actin served as controls. Relative signal intensity of KIF21B/actin ratios in %. n=3, each. 4h: wildtype (+/+) 100%, knockout (-/-) 100%; 8h: wildtype (+/+) 93.9±9.2, knockout (-/-) 110.4±12.9; 24h: wildtype (+/+) 57.8±11.5, knockout (-/-) 61.5±12.0; 48h: wildtype (+/+) 39.4±10.3, knockout (-/-) 35.4±9.6. (C, D) Relative signal intensities of KIF21B and KIF5 in hippocampal lysates remain equal across the genotypes (wildtype (+/+) versus <i>Trim3</i> knockout (-/-)). Relative signal intensity of KIF/NSE ratios in %. n=4 each. KIF21B: wildtype (+/+) 0.55±0.08, knockout (-/-) 0.69±0.03; KIF5: wildtype (+/+) 0.70±0.07, knockout (-/-): 0.72±0.03. ns: not significant. (E, F) Overexpression of TRIM3 does not alter endogenous KIF21B protein levels. Cultured hippocampal neurons (DIV10) were transfected with vectors encoding HA-TRIM3 or HA, respectively. Coexpression of GFP served as transfection control and volume marker. Cells were fixed and stained for endogenous KIF21B at DIV14. Somatic KIF21B signal intensity: HA-control: set to 100%, n= 40; HA-TRIM3: 104±17%, n=38. (Scale bars in E: 20 µm.) Data: means±SEM.</p

Subcellular localization of KIF21B and TRIM3.

<p>(A) KIF21B locates to the somato-dendritic compartment of neurons and is prominent in growth cones of young neurons (DIV7) (arrows). (Scale bar: 20 µm.) (B) Electron microscopy analysis, immunogold signals. TRIM3 locates close to the membrane of putative transport vesicles in neurons derived from hippocampal slices (arrow) (Scale bar: 50 nm.). (C) Electron microscopy analysis, DAB signals. TRIM3 locates to vesicles at the Golgi apparatus (arrows). M: mitochondria, Golgi: Golgi apparatus (Scale bar: 200 nm). (D, E) TRIM3 (red, left) or KIF21B (red, right) colocalize with MAP2-positive dendrites (green, arrows) but are not detected in GFAP-positive astrocytes (blue, arrowheads). (Scale bars: 20 µm.) (F) KIF21B and TRIM3 colocalize in punctate structures (yellow, arrowheads) across neuronal dendrites. (Scale bar: 20 µm, scale bar boxed region: 5 µm.).</p