The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

Genes implicated in translation control have been associated with autism spectrum disorders (ASDs). However, some important genetic causes of autism, including the 16p11.2 microdeletion, bear no obvious connection to translation. Here, we use proteomics, genetics, and translation assays in cultured cells and mouse brain to reveal altered translation mediated by loss of the kinase TAOK2 in 16p11.2 deletion models. We show that TAOK2 associates with the translational machinery and functions as a translational brake by phosphorylating eukaryotic elongation factor 2 (eEF2). Previously, all signal-mediated regulation of translation elongation via eEF2 phosphorylation was believed to be mediated by a single kinase, eEF2K. However, we show that TAOK2 can directly phosphorylate eEF2 on the same regulatory site, but functions independently of eEF2K signaling. Collectively, our results reveal an eEF2K-independent signaling pathway for control of translation elongation and suggest altered translation as a molecular component in the etiology of some forms of ASD.

Protocol for differential multi-omic analyses of distinct cell types in the mouse cerebral cortex.

Here, we present a protocol for differential multi-omic analyses of distinct cell types in the developing mouse cerebral cortex. We describe steps for in utero electroporation, subsequent flow-cytometry-based isolation of developing mouse cortical cells, bulk RNA sequencing or quantitative liquid chromatography-tandem mass spectrometry, and bioinformatic analyses. This protocol can be applied to compare the proteomes and transcriptomes of developing mouse cortical cell populations after various manipulations (e.g., epigenetic). For complete details on the use and execution of this protocol, please refer to Meka et al. (2022).1.

Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease-relevant pathologies.

There are hundreds of risk genes associated with autism spectrum disorder (ASD), but signaling networks at the protein level remain unexplored. We use neuron-specific proximity-labeling proteomics (BioID2) to identify protein-protein interaction (PPI) networks for 41 ASD risk genes. Neuron-specific PPI networks, including synaptic transmission proteins, are disrupted by de novo missense variants. The PPI network map reveals convergent pathways, including mitochondrial/metabolic processes, Wnt signaling, and MAPK signaling. CRISPR knockout displays an association between mitochondrial activity and ASD risk genes. The PPI network shows an enrichment of 112 additional ASD risk genes and differentially expressed genes from postmortem ASD patients. Clustering of risk genes based on PPI networks identifies gene groups corresponding to clinical behavior score severity. Our data report that cell type-specific PPI networks can identify individual and convergent ASD signaling networks, provide a method to assess patient variants, and highlight biological insight into disease mechanisms and sub-cohorts of ASD.

TAOK2 rescues autism-linked developmental deficits in a 16p11.2 microdeletion mouse model.

The precise development of the neocortex is a prerequisite for higher cognitive and associative functions. Despite numerous advances that have been made in understanding neuronal differentiation and cortex development, our knowledge regarding the impact of specific genes associated with neurodevelopmental disorders on these processes is still limited. Here, we show that Taok2, which is encoded in humans within the autism spectrum disorder (ASD) susceptibility locus 16p11.2, is essential for neuronal migration. Overexpression of de novo mutations or rare variants from ASD patients disrupts neuronal migration in an isoform-specific manner. The mutated TAOK2α variants but not the TAOK2ß variants impaired neuronal migration. Moreover, the TAOK2α isoform colocalizes with microtubules. Consequently, neurons lacking Taok2 have unstable microtubules with reduced levels of acetylated tubulin and phosphorylated JNK1. Mice lacking Taok2 develop gross cortical and cortex layering abnormalities. Moreover, acute Taok2 downregulation or Taok2 knockout delayed the migration of upper-layer cortical neurons in mice, and the expression of a constitutively active form of JNK1 rescued these neuronal migration defects. Finally, we report that the brains of the Taok2 KO and 16p11.2 del Het mouse models show striking anatomical similarities and that the heterozygous 16p11.2 microdeletion mouse model displayed reduced levels of phosphorylated JNK1 and neuronal migration deficits, which were ameliorated upon the introduction of TAOK2α in cortical neurons and in the developing cortex of those mice. These results delineate the critical role of TAOK2 in cortical development and its contribution to neurodevelopmental disorders, including ASD.

Centrosome-dependent microtubule modifications set the conditions for axon formation.

Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.

Emerging roles of the centrosome in neuronal development.

The role of the centrosome-a microtubule-organizing center-in neuronal development has been under scrutiny and is controversial. The function and position of the centrosome have been shown to play an important role in selecting the position of axon outgrowth in cultured neurons and in situ. However, other studies have shown that axonal growth is independent of centrosomal functions. Recent discoveries define the centrosome as an F-actin organizing organelle in various cell types; thus, giving a whole new perspective to the role of the centrosome in lymphocyte polarity, cell division, and neuronal development. These discoveries compel the need to revisit centrosomal functions by investigating the fundamental mechanisms that regulate centrosomal F-actin remodeling during neuronal differentiation and polarization. In this review, we summarize the up-to-date knowledge regarding the function of the centrosome in neuronal differentiation. We put special emphasis on recent findings describing the centrosome as an F-actin organizing center. Additionally, with the available data regarding centrosome, microtubules and F-actin organization, we provide a model on how centrosomal F-actin could be modulating neuronal differentiation and polarity.

Parkin deficiency perturbs striatal circuit dynamics.

Loss-of-function mutations in the parkin-encoding PARK2 gene are a frequent cause of young-onset, autosomal recessive Parkinson's disease (PD). Parkin knockout mice have no nigro-striatal neuronal loss but exhibit abnormalities of striatal dopamine transmission and cortico-striatal synaptic function. How these predegenerative changes observed in vitro affect neural dynamics at the intact circuit level, however, remains hitherto elusive. Here, we recorded from motor cortex, striatum and globus pallidus (GP) of anesthetized parkin-deficient mice to assess cortex-basal ganglia circuit dynamics and to dissect cell type-specific functional connectivity in the presymptomatic phase of genetic PD. While ongoing activity of presumed striatal spiny projection neurons and their downstream counterparts in the GP was not different from controls, parkin deficiency had a differential impact on striatal interneurons: In parkin-mutant mice, tonically active neurons displayed elevated activity levels. Baseline firing rates of transgenic striatal fast spiking interneurons (FSI), on the contrary, were reduced and the correlational structure of the FSI microcircuitry was disrupted. The entire transgenic striatal microcircuit showed enhanced and phase-shifted phase coupling to slow (1-3 Hz) cortical population oscillations. Unexpectedly, local field potentials recorded from striatum and GP of parkin-mutant mice robustly displayed amplified beta oscillations (~22 Hz), phase-coupled to cortex. Parkin deficiency selectively increased spike-field coupling of FSIs to beta oscillations. Our findings suggest that loss of parkin function leads to amplifications of synchronized cortico-striatal oscillations and an intrastriatal reconfiguration of interneuronal circuits. This presymptomatic disarrangement of dynamic functional connectivity may precede nigro-striatal neurodegeneration and predispose to imbalance of striatal outflow accompanying symptomatic PD.

Radial somatic F-actin organization affects growth cone dynamics during early neuronal development.

The centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center, raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here, we report, using super-resolution microscopy and live-cell imaging of cultured rodent neurons, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively. Photoactivation/photoconversion experiments and molecular manipulations of F-actin stability reveal a robust flux of somatic F-actin toward the cell periphery. Finally, we show that somatic F-actin intermingles with centrosomal PCM-1 (pericentriolar material 1 protein) satellites. Knockdown of PCM-1 and disruption of centrosomal activity not only affect F-actin dynamics near the centrosome but also in distal growth cones. Collectively, the data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers, hence sustaining initial neuronal development.

Microtubules Modulate F-actin Dynamics during Neuronal Polarization.

Neuronal polarization is reflected by different dynamics of microtubule and filamentous actin (F-actin). Axonal microtubules are more stable than those in the remaining neurites, while dynamics of F-actin in axonal growth cones clearly exceed those in their dendritic counterparts. However, whether a functional interplay exists between the microtubule network and F-actin dynamics in growing axons and whether this interplay is instrumental for breaking cellular symmetry is currently unknown. Here, we show that an increment on microtubule stability or number of microtubules is associated with increased F-actin dynamics. Moreover, we show that Drebrin E, an F-actin and microtubule plus-end binding protein, mediates this cross talk. Drebrin E segregates preferentially to growth cones with a higher F-actin treadmilling rate, where more microtubule plus-ends are found. Interruption of the interaction of Drebrin E with microtubules decreases F-actin dynamics and arrests neuronal polarization. Collectively the data show that microtubules modulate F-actin dynamics for initial axon extension during neuronal development.

DIXDC1 Phosphorylation and Control of Dendritic Morphology Are Impaired by Rare Genetic Variants.

The development of neural connectivity is essential for brain function, and disruption of this process is associated with autism spectrum disorders (ASDs). DIX domain containing 1 (DIXDC1) has previously been implicated in neurodevelopmental disorders, but its role in postnatal brain function remains unknown. Using a knockout mouse model, we determined that DIXDC1 is a regulator of excitatory neuron dendrite development and synapse function in the cortex. We discovered that MARK1, previously linked to ASDs, phosphorylates DIXDC1 to regulate dendrite and spine development through modulation of the cytoskeletal network in an isoform-specific manner. Finally, rare missense variants in DIXDC1 were identified in ASD patient cohorts via genetic sequencing. Interestingly, the variants inhibit DIXDC1 isoform 1 phosphorylation, causing impairment to dendrite and spine growth. These data reveal that DIXDC1 is a regulator of cortical dendrite and synaptic development and provide mechanistic insight into morphological defects associated with neurodevelopmental disorders.

Parkin cooperates with GDNF/RET signaling to prevent dopaminergic neuron degeneration.

Parkin and the glial cell line-derived neurotrophic factor (GDNF) receptor RET have both been independently linked to the dopaminergic neuron degeneration that underlies Parkinson's disease (PD). In the present study, we demonstrate that there is genetic crosstalk between parkin and the receptor tyrosine kinase RET in two different mouse models of PD. Mice lacking both parkin and RET exhibited accelerated dopaminergic cell and axonal loss compared with parkin-deficient animals, which showed none, and RET-deficient mice, in which we found moderate degeneration. Transgenic expression of parkin protected the dopaminergic systems of aged RET-deficient mice. Downregulation of either parkin or RET in neuronal cells impaired mitochondrial function and morphology. Parkin expression restored mitochondrial function in GDNF/RET-deficient cells, while GDNF stimulation rescued mitochondrial defects in parkin-deficient cells. In both cases, improved mitochondrial function was the result of activation of the prosurvival NF-κB pathway, which was mediated by RET through the phosphoinositide-3-kinase (PI3K) pathway. Taken together, these observations indicate that parkin and the RET signaling cascade converge to control mitochondrial integrity and thereby properly maintain substantia nigra pars compacta dopaminergic neurons and their innervation in the striatum. The demonstration of crosstalk between parkin and RET highlights the interplay in the protein network that is altered in PD and suggests potential therapeutic targets and strategies to treat PD.

Redox activated MAP kinase death signaling cascade initiated by ASK1 is not activated in female mice following MPTP: novel mechanism of neuroprotection.

Incidence of Parkinson's disease (PD) is lower in women compared to men (1:1.46), which is reflected in animal models. However, precise mechanisms are unclear. Administration of MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) to female mice does not lead to mitochondrial complex I inhibition as seen in males and the progressive dopaminergic cell loss in substantia nigra (SNpc) is significantly attenuated. Redox driven apoptotic signaling pathways regulated by thiol disulfide oxidoreductase(s) have been implicated in the neurodegeneration seen in PD. Oxidation of thioredoxin leads to activation of apoptosis signal regulating kinase 1 (ASK1; MAPKKK) initiating cell death cascade through MAP kinase(s). Higher constitutive expression of enzymes involved in cellular redox maintenance, such as glutathione reductase, thioredoxin, and thioredoxin reductase is observed in female brain. Exposure to MPTP activates ASK1 in male but not in female mice. Higher expression of Trx in females potentially prevents ASK1 activation. Downstream of ASK1, phosphorylation of p38 MAP kinase is seen in male but not female mice. Expression of DJ-1, the redox sensing protein is higher in females and the loss of nuclear DJ-1, followed by translocation of Daxx (death associated protein) from the nucleus to the cytosol, which promotes ASK1 mediated death cascade is not seen in females. The enzymes involved in redox maintenance potentially could play a crucial role in preventing the activation of redox driven death signaling cascade and offer neuroprotection. Theraupeutic strategies that help maintain redox homeostasis may help prevent the progressive neurodegeneration seen in PD.

Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of substantia nigra leads to nuclear translocation of p53 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Activation of the mixed lineage kinase and c-Jun N-terminal kinase (JNK) has been reported in models of PD. Our focus was to discern whether distinct pathways were activated in cell-specific manner within the SNpc. We now demonstrate the selective phosphorylation of p38 MAP kinase within the dopaminergic neurons, whereas JNK activation occurs predominantly in the microglia. p38 activation results in downstream phosphorylation of p53 and increased p53 mediated transcription of Bax and Puma in the ventral midbrain. Treatment with p38 inhibitor, SB239063 protected primary dopaminergic neurons derived from human progenitor cells from MPP(+) mediated cell death and prevented the downstream phosphorylation of p53 and its translocation to the nucleus in vivo, in the ventral midbrain. The increased staining of phosphorylated p38 in the surviving neurons of SNpc in human brain sections from patients with PD and in MPTP treated mice but not in the ventral tegmental area provides further evidence suggesting a role for p38 in the degeneration of dopaminergic neurons of SNpc. We thus demonstrate the cell specific activation of MAP kinase pathways within the SNpc after MPTP treatment emphasizing the role of multiple signaling cascades in the pathogenesis and progression of the disease. Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression.