Functional Inhibition of Katanin Affects Synaptic Plasticity.

Dynamic microtubules critically regulate synaptic functions, but the role of microtubule severing in these processes is barely understood. Katanin is a neuronally expressed microtubule-severing complex regulating microtubule number and length in cell division or neurogenesis; however, its potential role in synaptic functions has remained unknown. Studying mice from both sexes, we found that katanin is abundant in neuronal dendrites and can be detected at individual excitatory spine synapses. Overexpression of a dominant-negative ATPase-deficient katanin subunit to functionally inhibit severing alters the growth of microtubules in dendrites, specifically at premature but not mature neuronal stages without affecting spine density. Notably, interference with katanin function prevented structural spine remodeling following single synapse glutamate uncaging and significantly affected the potentiation of AMPA-receptor-mediated excitatory currents after chemical induction of long-term potentiation. Furthermore, katanin inhibition reduced the invasion of microtubules into fully developed spines. Our data demonstrate that katanin-mediated microtubule severing regulates structural and functional plasticity at synaptic sites.

Tetraspanin 15 depletion impairs extracellular vesicle docking at target neurons.

Neurons in the central nervous system release extracellular vesicles (EVs) and exosomes in response to synaptic activity to regulate physiological processes at target neurons. The intercellular transfer of proteins, mRNAs, lipids or metabolites through EVs potentially modulates the structure and function of neurons and circuits. Whereas the biogenesis of EVs, their release from donor cells, and their molecular composition have been studied extensively, the critical factors and mechanisms regulating EV interactions with target cells are incompletely understood. Here, we identified tetraspanin 15 (Tspan15) as a component of tumor susceptibility gene 101 protein (TSG101)- and CD81-positive EV fractions. Tspan15 fluorescent fusion proteins were released from donor cells and interacted with target cells together with the exosomal marker CD63. EVs collected from wildtype cortical neurons (WT-EVs) underwent similar association with target neurons derived from either wildtype (+/+) or Tspan15 knockout (-/-) mice. In contrast, target cell interactions of EVs collected from Tspan15 (-/-) cortical donor neurons (KO-EVs) were significantly impaired, as compared to WT-EVs. Our data suggest that Tspan15 is dispensable at target neuron plasma membranes, but is required at the EV surface to promote EV docking at target neurons.

Alpha- and beta-tubulin isotypes are differentially expressed during brain development.

Alpha- and beta-tubulin dimers polymerize into protofilaments that associate laterally to constitute a hollow tube, the microtubule. A dynamic network of interlinking filaments forms the microtubule cytoskeleton, which maintains the structure of cells and is key to various cellular processes including cell division, cell migration, and intracellular transport. Individual microtubules have an identity that depends on the differential integration of specific alpha- and beta-tubulin isotypes and is further specified by a variety of posttranslational modifications (PTMs). It is barely understood to which extent neighboring microtubules differ in their tubulin composition or whether specific tubulin isotypes cluster along the polymer. Furthermore, our knowledge about the spatio-temporal expression patterns of tubulin isotypes is limited, not at least due to the lack of antibodies or antibody cross-reactivities. Here, we asked which alpha- and beta-tubulin mRNAs and proteins are expressed in developing hippocampal neuron cultures and ex vivo brain tissue lysates. Using heterologous expression of GFP-tubulin fusion proteins, we systematically tested antibody-specificities against various tubulin isotypes. Our data provide quantitative information about tubulin expression levels in the mouse brain and classify tubulin isotypes during pre- and postnatal development.

Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits.

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

The Microtubule Severing Protein Katanin Regulates Proliferation of Neuronal Progenitors in Embryonic and Adult Neurogenesis.

Microtubule severing regulates cytoskeletal rearrangement underlying various cellular functions. Katanin, a heterodimer, consisting of catalytic (p60) and regulatory (p80) subunits severs dynamic microtubules to modulate several stages of cell division. The role of p60 katanin in the mammalian brain with respect to embryonic and adult neurogenesis is poorly understood. Here, we generated a Katna1 knockout mouse and found that consistent with a critical role of katanin in mitosis, constitutive homozygous Katna1 depletion is lethal. Katanin p60 haploinsufficiency induced an accumulation of neuronal progenitors in the subventricular zone during corticogenesis, and impaired their proliferation in the adult hippocampus dentate gyrus (DG) subgranular zone. This did not compromise DG plasticity or spatial and contextual learning and memory tasks employed in our study, consistent with the interpretation that adult neurogenesis may be associated with selective forms of hippocampal-dependent cognitive processes. Our data identify a critical role for the microtubule-severing protein katanin p60 in regulating neuronal progenitor proliferation in vivo during embryonic development and adult neurogenesis.

Myosin XVI Regulates Actin Cytoskeleton Dynamics in Dendritic Spines of Purkinje Cells and Affects Presynaptic Organization.

The actin cytoskeleton is crucial for function and morphology of neuronal synapses. Moreover, altered regulation of the neuronal actin cytoskeleton has been implicated in neuropsychiatric diseases such as autism spectrum disorder (ASD). Myosin XVI is a neuronally expressed unconventional myosin known to bind the WAVE regulatory complex (WRC), a regulator of filamentous actin (F-actin) polymerization. Notably, the gene encoding the myosin's heavy chain (MYO16) shows genetic association with neuropsychiatric disorders including ASD. Here, we investigated whether myosin XVI plays a role for actin cytoskeleton regulation in the dendritic spines of cerebellar Purkinje cells (PCs), a neuronal cell type crucial for motor learning, social cognition and vocalization. We provide evidence that both myosin XVI and the WRC component WAVE1 localize to PC spines. Fluorescence recovery after photobleaching (FRAP) analysis of GFP-actin in cultured PCs shows that Myo16 knockout as well as PC-specific Myo16 knockdown, lead to faster F-actin turnover in the dendritic spines of PCs. We also detect accelerated F-actin turnover upon interference with the WRC, and upon inhibition of Arp2/3 that drives formation of branched F-actin downstream of the WRC. In contrast, inhibition of formins that are responsible for polymerization of linear actin filaments does not cause faster F-actin turnover. Together, our data establish myosin XVI as a regulator of the postsynaptic actin cytoskeleton and suggest that it is an upstream activator of the WRC-Arp2/3 pathway in PC spines. Furthermore, ultra-structural and electrophysiological analyses of Myo16 knockout cerebellum reveals the presence of reduced numbers of synaptic vesicles at presynaptic terminals in the absence of the myosin. Therefore, we here define myosin XVI as an F-actin regulator important for presynaptic organization in the cerebellum.

Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression.

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

Growth Arrest Specific 6 Concentration is Increased in the Cerebrospinal Fluid of Patients with Alzheimer's Disease.

Growth arrest specific 6 (Gas6) has neurotrophic and neuroinflammatory functions, and may play a role in Alzheimer's disease (AD). In keeping with this hypothesis, we observed that cerebrospinal fluid (CSF) Gas6 is increased in AD patients compared to controls (63 versus 67 subjects; median value 13.3 versus 9.1 ng/ml; p < 0.0001). Thereafter, we assessed whether CSF Gas6 concentration was correlated to the following parameters: disease duration, MMSE score two years after clinical diagnosis, AD CSF biomarkers, and years of formal schooling. We detected an inverse correlation between CSF Gas6 levels at diagnosis and both disease duration (p < 0.0001) and decrease in the MMSE score two years later (p < 0.0001). Conversely, we found no correlation between CSF Gas6 and both AD biomarkers and years of formal schooling. In conclusion, our results suggest that upregulation of CSF Gas6 may be part of a defensive response aimed at counteracting AD progression.

APP is cleaved by Bace1 in pre-synaptic vesicles and establishes a pre-synaptic interactome, via its intracellular domain, with molecular complexes that regulate pre-synaptic vesicles functions.

Amyloid Precursor Protein (APP) is a type I membrane protein that undergoes extensive processing by secretases, including BACE1. Although mutations in APP and genes that regulate processing of APP, such as PSENs and BRI2/ITM2B, cause dementias, the normal function of APP in synaptic transmission, synaptic plasticity and memory formation is poorly understood. To grasp the biochemical mechanisms underlying the function of APP in the central nervous system, it is important to first define the sub-cellular localization of APP in synapses and the synaptic interactome of APP. Using biochemical and electron microscopy approaches, we have found that APP is localized in pre-synaptic vesicles, where it is processed by Bace1. By means of a proteomic approach, we have characterized the synaptic interactome of the APP intracellular domain. We focused on this region of APP because in vivo data underline the central functional and pathological role of the intracellular domain of APP. Consistent with the expression of APP in pre-synaptic vesicles, the synaptic APP intracellular domain interactome is predominantly constituted by pre-synaptic, rather than post-synaptic, proteins. This pre-synaptic interactome of the APP intracellular domain includes proteins expressed on pre-synaptic vesicles such as the vesicular SNARE Vamp2/Vamp1 and the Ca2+ sensors Synaptotagmin-1/Synaptotagmin-2, and non-vesicular pre-synaptic proteins that regulate exocytosis, endocytosis and recycling of pre-synaptic vesicles, such as target-membrane-SNAREs (Syntaxin-1b, Syntaxin-1a, Snap25 and Snap47), Munc-18, Nsf, α/ß/γ-Snaps and complexin. These data are consistent with a functional role for APP, via its carboxyl-terminal domain, in exocytosis, endocytosis and/or recycling of pre-synaptic vesicles.

Intensive rehabilitation increases BDNF serum levels in parkinsonian patients: a randomized study.

BACKGROUND: Exercise may decrease the risk of Parkinson's disease (PD) in humans and reduce PD symptoms in animal models. The beneficial effects have been linked to increased levels of neurotrophic factors. OBJECTIVE: We examined whether intensive rehabilitation treatment reduces motor disability in patients in the early stages of PD and increases brain-derived neurotrophic factor (BDNF) serum levels. METHODS: Thirty participants in the early stages of PD treated with rasagiline were randomly assigned to 3 hours of rehabilitation treatment that included aerobic exercise for 28 days (Group 1) or to not therapy (control; Group 2). BDNF serum levels were assessed at time T0 (baseline, before treatment), T1 (10 days), T2 (20 days), and T3 (28 days). At T0 and T3, we assessed the Unified Parkinson's Disease Rating Scale (UPDRS) III in both groups, as well as the UPDRS II and total, Berg Balance Scale, and 6-minute walking test only in Group 1. RESULTS: BDNF levels significantly increased at T1 in Group 1, an increase that was maintained throughout the treatment period. At T3 compared to T0, UPDRS III scores significantly improved in Group 1 along with scores for UPDRS II, total, Berg Balance Scale, and 6-minute walking test. CONCLUSIONS: Intensive rehabilitation treatment increases the BDNF levels and improves PD signs in patients in the early stages of the disease. These results are in line with studies on animal models of PD and healthy subjects.

An intracellular threonine of amyloid-ß precursor protein mediates synaptic plasticity deficits and memory loss.

Mutations in Amyloid-ß Precursor Protein (APP) and BRI2/ITM2b genes cause Familial Alzheimer and Danish Dementias (FAD/FDD), respectively. APP processing by BACE1, which is inhibited by BRI2, yields sAPPß and ß-CTF. ß-CTF is cleaved by gamma-secretase to produce Aß. A knock-in mouse model of FDD, called FDDKI, shows deficits in memory and synaptic plasticity, which can be attributed to sAPPß/ß-CTF but not Aß. We have investigated further the pathogenic function of ß-CTF focusing on Thr(668) of ß-CTF because phosphorylation of Thr(668) is increased in AD cases. We created a knock-in mouse bearing a Thr(668)Ala mutation (APP(TA) mice) that prevents phosphorylation at this site. This mutation prevents the development of memory and synaptic plasticity deficits in FDDKI mice. These data are consistent with a role for the carboxyl-terminal APP domain in the pathogenesis of dementia and suggest that averting the noxious role of Thr(668) is a viable therapeutic strategy for human dementias.

Tyr682 in the Aß-precursor protein intracellular domain regulates synaptic connectivity, cholinergic function, and cognitive performance.

Processing of Aß-precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the 682YENPTY687 motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Y682 to Gly (APP(YG/YG) mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP-like protein 2 KO background. This evidence prompted us to characterize further the APP(YG/YG) mice. Here, we show that APP(YG/YG) mice develop aging-dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP(YG/YG) mice. The profound deficits caused by the Y682 mutation underscore the biological importance of APP and indicate that APP(YG/YG) are a valuable mouse model to study APP functions in physiological and pathological processes.