Regulation of Dyenin-Dependent Neuronal Transport and Trafficking by Cdk5

Neurons have a distinct structure; they are the only cells in the body whose proximal and distal ends can be separated by more than a meter. This leads to unique challenges for the neuron, specifically, how proteins, organelles, and other cargo synthesized in the cell body are specifically trafficked from the soma to the distal end of the axon, and how degradative cargo and signaling factors are transported from the terminal to the cell body. Using primary cultured central and peripheral neurons isolated from the brains and spinal cords of rats or mice, we sought to determine if a neuronal-specific kinase, cyclin dependent kinase 5 (CDK5) regulates the motility of cargo moving along the axon, and if this kinase also regulates the localized exclusion of somatodendritic cargo from the axon. Within the mid-axon, we observed that baseline CDK5 activity was not required to regulate axonal transport, but pathological activation of CDK5 via a stress-associated activator disrupted both anterograde (outward) and retrograde (inward) motility. In contrast, within the axon initial segment (AIS), inhibition of normal CDK5 activity disrupted cytoskeletal structure and compromised axonal and dendritic sorting, aberrantly permitting the entry of somatodendritic cargos into the axon. We determined that both roles of CDK5 in axonal regulation were dependent on phosphorylation of target Ndel1, a protein that regulates the interaction of the retrograde microtubule-based motor dynein with its cofactor Lis1. In the mid-axon, high levels of CDK5 activity causes dynein to tightly bind along the microtubule interrupting processive motility, while in the AIS, CDK5 activity is required to initiate dynein-driven return of somatodendritic cargo to the cell body. Together theses studies demonstrate the importance of CDK5 activity in the regulation of transport within the neuron

Novel SGCE mutation in a patient with myoclonus-dystonia: A case report

OBJECTIVES: Characterize the presentation, workup, and management of METHODS: A woman with myoclonus and dystonia was identified based on clinical history and physical examination. Workup was conducted to determine the cause of her symptoms, including whole-exome sequencing. Myoclonus-dystonia is associated with more than 100 distinct mutations in MYC/DYT RESULTS: Childhood onset myoclonus and worsening dystonia with age were identified in a young woman. She underwent screening for common causes of twitching movements, followed by whole-exome sequencing which identified a de novo novel variant in the DISCUSSION: Myoclonus-dystonia should be considered in patients with symptoms of head and upper extremity myoclonus early in life, especially with co-occurring dystonia, even in the absence of a family history of similar symptoms. Diagnosis of this condition should take place using sequencing, as new mutations continue to be discovered

Regulation of dynein-dependent neuronal transport and trafficking by CDK5

Neurons have a distinct structure; they are the only cells in the body whose proximal and distal ends can be separated by more than a meter. This leads to unique challenges for the neuron, specifically, how proteins, organelles, and other cargo synthesized in the cell body are specifically trafficked from the soma to the distal end of the axon, and how degradative cargo and signaling factors are transported from the terminal to the cell body. Using primary cultured central and peripheral neurons isolated from the brains and spinal cords of rats or mice, we sought to determine if a neuronal-specific kinase, cyclin dependent kinase 5 (CDK5) regulates the motility of cargo moving along the axon, and if this kinase also regulates the localized exclusion of somatodendritic cargo from the axon. Within the mid-axon, we observed that baseline CDK5 activity was not required to regulate axonal transport, but pathological activation of CDK5 via a stress-associated activator disrupted both anterograde (outward) and retrograde (inward) motility. In contrast, within the axon initial segment (AIS), inhibition of normal CDK5 activity disrupted cytoskeletal structure and compromised axonal and dendritic sorting, aberrantly permitting the entry of somatodendritic cargos into the axon. We determined that both roles of CDK5 in axonal regulation were dependent on phosphorylation of target Ndel1, a protein that regulates the interaction of the retrograde microtubule-based motor dynein with its cofactor Lis1. In the mid-axon, high levels of CDK5 activity causes dynein to tightly bind along the microtubule interrupting processive motility, while in the AIS, CDK5 activity is required to initiate dynein-driven return of somatodendritic cargo to the cell body. Together theses studies demonstrate the importance of CDK5 activity in the regulation of transport within the neuron

Stress-Induced CDK5 Activation Disrupts Axonal Transport via Lis1/Ndel1/Dynein

SummaryAxonal transport is essential for neuronal function, and defects in transport are associated with multiple neurodegenerative diseases. Aberrant cyclin-dependent kinase 5 (CDK5) activity, driven by the stress-induced activator p25, also is observed in these diseases. Here we show that elevated CDK5 activity increases the frequency of nonprocessive events for a range of organelles, including lysosomes, autophagosomes, mitochondria, and signaling endosomes. Transport disruption induced by aberrant CDK5 activation depends on the Lis1/Ndel1 complex, which directly regulates dynein activity. CDK5 phosphorylation of Ndel1 favors a high affinity Lis1/Ndel/dynein complex that blocks the ATP-dependent release of dynein from microtubules, inhibiting processive motility of dynein-driven cargo. Similar transport defects observed in neurons from a mouse model of amyotrophic lateral sclerosis are rescued by CDK5 inhibition. Together, these studies identify CDK5 as a Lis1/Ndel1-dependent regulator of transport in stressed neurons, and suggest that dysregulated CDK5 activity contributes to the transport deficits observed during neurodegeneration

Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-β, and TGF-α is specifically triggered by different stimuli and involves different PKC isoenzymes

Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-α), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage—hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]—are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS) -based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-α. HB-EGF, NRG, and TGF-α cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-α was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-α and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different extracellular stimuli into ectodomain cleavage.—Herrlich, A., Klinman, E., Fu, J., Sadegh, C., Lodish, H. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-β, and TGF-α is specifically triggered by different stimuli and involves different PKC isoenzymes

Molecular Neuropathology in Practice: Clinical Profiling and Integrative Analysis of Molecular Alterations in Glioblastoma

Molecular profiling of glioblastoma has revealed complex cytogenetic, epigenetic, and molecular abnormalities that are necessary for diagnosis, prognosis, and treatment. Our neuro-oncology group has developed a data-driven, institutional consensus guideline for efficient and optimal workup of glioblastomas based on our routine performance of molecular testing. We describe our institution’s testing algorithm, assay development, and genetic findings in glioblastoma, to illustrate current practices and challenges in neuropathology related to molecular and genetic testing. We have found that coordination of test requisition, tissue handling, and incorporation of results into the final pathologic diagnosis by the neuropathologist improve patient care. Here, we present analysis of O 6 -methylguanine-DNA-methyltransferase promoter methylation and next-generation sequencing results of 189 patients, obtained utilizing our internal processes led by the neuropathology team. Our institutional pathway for neuropathologist-driven molecular testing has streamlined the management of glioblastoma samples for efficient return of results for incorporation of genomic data into the pathological diagnosis and optimal patient care