The MAP She1 coordinates directional spindle positioning by spatially restricting dynein activity

Dynein motors move the mitotic spindle to the cell division plane in many cell types, including in budding yeast, in which dynein is assisted by numerous factors including the microtubule-associated protein (MAP) She1. Evidence suggests that She1 plays a role in polarizing dynein-mediated spindle movements toward the daughter cell; however, how She1 performs this function is unknown. We find that She1 assists dynein in maintaining the spindle in close proximity to the bud neck, such that at anaphase onset the chromosomes are segregated to mother and daughter cells. She1 does so by attenuating the initiation of dynein-mediated spindle movements within the mother cell, thus ensuring such movements are polarized toward the daughter cell. Our data indicate that this activity relies on She1 binding to the microtubule-bound conformation of the dynein microtubule-binding domain, and to astral microtubules within mother cells. Our findings reveal how an asymmetrically localized MAP directionally tunes dynein activity by attenuating motor activity in a spatially confined manner.

Dynamic interactions and Ca2+-binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid β aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies.

Dysregulated coordination of MAPT exon 2 and exon 10 splicing underlies different tau pathologies in PSP and AD

ABSTRACTUnderstanding regulation of MAPT splicing is important to the etiology of many nerurodegenerative diseases, including Alzheimer disease (AD) and progressive supranuclear palsy (PSP), in which different tau isoforms accumulate in pathologic inclusions. MAPT, the gene encoding the tau protein, undergoes complex alternative pre-mRNA splicing to generate six isoforms. Tauopathies can be categorized by the presence of tau aggregates containing either 3 (3R) or 4 (4R) microtubule binding domain repeats (determined by inclusion/exclusion of exon 10), but the role of the N terminal domain of the protein, determined by inclusion/exclusion of exons 2 and 3 has been less well studied. Using an unbiased correlational screen in human brain tissue, we observed coordination of MAPT exons 2 and 10 splicing. Expression of exon 2 splicing regulators and subsequently exon 2 inclusion are differentially disrupted in PSP and AD brain, resulting in the accumulation of 1N4R isoforms in PSP and 0N isoforms in AD temporal cortex. Furthermore, we identified different N-terminal isoforms of tau present in neurofibrillary tangles, dystrophic neurites and tufted astrocytes, indicating a role for differential N-terminal splicing in the development of disparate tau neuropathologies. We conclude that N-terminal splicing and combinatorial regulation with exon 10 inclusion/exclusion is likely to be important to our understanding of tauopathies.

Tau Isoform Profile in Essential Tremor Diverges From Other Tauopathies

Patients with essential tremor (ET) frequently develop concurrent dementia, which is often assumed to represent co-morbid Alzheimer disease (AD). Autopsy studies have identified a spectrum of tau pathologies in ET and tau isoforms have not been examined in ET. We performed immunoblotting using autopsy cerebral cortical tissue from patients with ET (n = 13), progressive supranuclear palsy ([PSP], n = 10), Pick disease ([PiD], n = 2), and AD (n = 7). Total tau in ET samples was similar to that in PSP and PiD but was significantly lower than that in AD. Abnormal tau levels measured using the AT8 phospho-tau specific (S202/T205/S208) monoclonal antibody in ET were similar to those in PSP but were lower than in PiD and AD. In aggregates, tau with 3 microtubule-binding domain repeats (3R) was significantly higher in AD than ET, while tau with 4 repeats (4R) was significantly higher in PSP. Strikingly, the total tau without N-terminal inserts in ET was significantly lower than in PSP, PiD, and AD, but total tau with other N-terminal inserts was not. Monomeric tau with one insert in ET was similar to that in PSP and PiD was lower than in AD. Thus, ET brains exhibit an expression profile of tau protein isoforms that diverges from that of other tauopathies.

Autoregulatory control of microtubule binding in doublecortin-like kinase 1

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

Effect of conditional deletion of cytoplasmic dynein heavy chain DYNC1H1 on postnatal photoreceptors

Cytoplasmic dynein (dynein 1), a major retrograde motor of eukaryotic cells, is a 1.4 MDa protein complex consisting of a pair of heavy chains (DYNC1H1) and a set of heterodimeric noncatalytic accessory components termed intermediate, light intermediate and light chains. DYNC1H1 (4644 amino acids) is the dynein backbone encoded by a gene consisting of 77 exons. We generated a floxed Dync1h1 allele that excises exons 24 and 25 and truncates DYNC1H1 during Six3Cre-induced homologous recombination. Truncation results in loss of the motor and microtubule-binding domain. Dync1h1F/F;Six3Cre photoreceptors degenerated rapidly within two postnatal weeks. In the postnatal day 6 (P6) Dync1h1F/F;Six3Cre central retina, outer and inner nuclear layers were severely disorganized and lacked a recognizable outer plexiform layer (OPL). Although the gene was effectively silenced by P6, DYNC1H1 remnants persisted and aggregated together with rhodopsin, PDE6 and centrin-2-positive centrosomes in the outer nuclear layer. As photoreceptor degeneration is delayed in the Dync1h1F/F;Six3Cre retina periphery, retinal lamination and outer segment elongation are in part preserved. DYNC1H1 strongly persisted in the inner plexiform layer (IPL) beyond P16 suggesting lack of clearance of the DYNC1H1 polypeptide. This persistence of DYNC1H1 allows horizontal, rod bipolar, amacrine and ganglion cells to survive past P12. The results show that cytoplasmic dynein is essential for retina lamination, nuclear positioning, vesicular trafficking of photoreceptor membrane proteins and inner/outer segment elaboration.

The MAP She1 coordinates directional spindle positioning by spatially restricting dynein activity

ABSTRACTDynein motors move the mitotic spindle to the cell division plane in many cell types, including in budding yeast, in which dynein is assisted by numerous factors including the microtubule-associated protein (MAP) She1. Evidence suggests that She1 plays a role in polarizing dynein-mediated spindle movements toward the daughter cell; however, how She1 performs this function is unknown. We find that She1 assists dynein in maintaining the spindle close to the bud neck, such that at anaphase onset the chromosomes are segregated to mother and daughter cells. She1 does so by attenuating the initiation of dynein-mediated spindle movements specifically within the mother cell, ensuring such movements are polarized toward the daughter cell. Our data indicate that this activity relies on She1 binding to the microtubule-bound conformation of the dynein microtubule-binding domain, and to astral microtubules within mother cells. Our findings reveal how an asymmetrically localized MAP directionally tunes dynein activity by attenuating motor activity in a spatially confined manner.

In-frame deletion of SPECC1L microtubule binding domain results in embryonic tissue movement and fusion defects

Embryonic morphogenesis of the neural tube, palate, ventral body wall and optic fissure require precise sequence of tissue movement and fusion, which if incomplete, leads to anencephaly/exencephaly, cleft palate, omphalocele and coloboma, respectively. These are genetically heterogeneous birth defects, so there is a continued need to identify etiologic genes. Patients with autosomal dominant SPECC1L mutations show syndromic malformations, including hypertelorism, cleft palate and omphalocele. These SPECC1L mutations cluster in the second coiled-coil domain (CCD2), which facilitates association with microtubules. To study SPECC1L function in mice, we first generated a null allele (Specc1lΔEx4) lacking the entire SPECC1L protein. Homozygous mutants for these truncations died perinatally without cleft palate or exencephaly. Given the clustering of human mutations in CCD2, we hypothesized that targeted perturbation of CCD2 may be required. Indeed, homozygotes for in-frame deletions involving CCD2 (Specc1lΔCCD2) resulted in ~50% exencephaly and ~50% cleft palate. Interestingly, these two phenotypes are never observed in the same embryo. Examination of embryos with and without exencephaly revealed that the oral cavity was narrower in exencephalic embryos, which allowed palatal shelves to elevate despite their defect. In contrast to an evenly distributed subcellular expression pattern, mutant SPECC1L-ΔCCD2 protein showed abnormal subcellular localization, decreased overlap with microtubules, increased actin bundles, and dislocated non-muscle myosin II to the cell cortex. Thus, we show that perturbations of CCD2 in the context of full SPECC1L protein affects tissue fusion dynamics, indicating that human SPECC1L CCD2 mutations are gain-of-function. Improper SPECC1L subcellular localization appears to disrupt connections between actomyosin and microtubule networks, which in turn may affect cell alignment and coordinate movement during tissue morphogenesis.

Doxycycline interferes with tau amyloid aggregation abolishing its associated neuronal toxicity

Tauopathies are neurodegenerative disorders with increasing incidence and still without cure. The extensive time required for development and approval of novel therapeutics highlights the need for testing and repurposing known safe molecules. Since doxycycline impacts α-synuclein aggregation and toxicity, herein we tested its effect on tau. We found that doxycycline reduces amyloid aggregation of the different isoforms of tau protein in a dose-dependent manner, remodeling the resultant species. Furthermore, doxycycline interacts with tau microtubule-binding domain preventing its aggregation. In a cell free system doxycycline also prevents tau seeding and in cell culture reduces toxicity of tau aggregates. Overall, our results expand the spectrum of action of doxycycline against aggregation-prone proteins, opening novel perspectives for its repurposing as a disease-modifying drug for tauopathies.