Cytoskeletal mechanisms of neuronal morphogenesis

The cytoskeleton is the major intracellular structure that determines the morphology of a neuron. Thus, mechanisms that ensure a precisely regulated assembly of cytoskeletal elements in time and space have an important role in the development from a morphologically simple neuronal precursor cell to a complex polarized neuron that can establish contacts to several hundreds of other cells. Here, cytoskeletal mechanisms that underlie the formation of neurites, directed elongation and stabilization of neuronal processes are summarized. It has become evident that different cytoskeletal elements are highly crosslinked with each other by several classes of specific linker proteins. Of these, microtubule-associated proteins (MAPs) appear to have an important role in connecting the microtubule skeleton to other cytoskeletal filaments and plasma membrane components during neuronal morphogenesis. Future experiments will have to elucidate the function and the regulation of the neuronal cytoskeleton in an authentic nervous system environment during development. Recent approaches are discussed at the end of this article.     

Cytoskeletal changes in diseases of the nervous system

The neuronal cytoskeleton not only provides the structural backbone of neurons, but also plays a fundamental role in maintaining neuronal functions. Dysregulation of neuronal architecture is evident in both injury and diseases of the central nervous system. These changes often result in the disruption of protein trafficking, loss of synapses and the death of neurons, ultimately impacting on signal transmission and manifesting in the disease phenotype. Furthermore, mutations in cytoskeletal proteins have been implicated in numerous diseases and, in some cases, identified as the cause of the disease, highlighting the critical role of the cytoskeleton in disease pathology. This review focuses on the role of cytoskeletal proteins in the pathology of mental disorders, neurodegenerative diseases and motor function deficits. In particular, we illustrate how cytoskeletal proteins can be directly linked to disease pathology and progression.     

Cell death mechanisms in neurodegeneration

Progressive cell loss in specific neuronal populations often associated with typical cytoskeletal protein aggregations is a pathological hallmark of neurodegenerative disorders, but the nature, time course and molecular causes of cell death and their relation to cytoskeletal pathologies are still unresolved. Apoptosis or alternative pathways of cell death have been discussed in Alzheimer's disease and other neurodegenerative disorders. Apoptotic DNA fragmentation in human brain as a sign of neuronal injury is found too frequent as to account for continous neuron loss in these slowly progressive processes. Morphological studies revealed extremely rare apoptotic neuronal death in Alzheimer's disease but yielded mixed results for Parkinson's disease and other neurodegenerative disorders. Based on recent data in human brain, as well as in animal and cell culture models, a picture is beginning to emerge suggesting that, in addition to apoptosis, other forms of programmed cell death may participate in neurodegeneration. Better understanding of the molecular players will further elucidate the mechanisms of cell death in these disorders and their relations to cytoskeletal abnormalities. Susceptible cell populations in a proapoptotic environment show increased vulnerability towards multiple noxious factors discussed in the pathogenesis of neurodegeneration. In conclusion, although many in vivo and in vitro data are in favor of apoptosis involvement in neurodegenerative processes, there is considerable evidence that very complex events may contribute to neuronal death with possible repair mechanisms, the elucidation of which may prove useful for future prevention and therapy of neurodegenerative disorders.     

Motor neurons rely on motor proteins

The importance of active axonal transport to the neuron has been highlighted by the recent discoveries that mutations in microtubule motor proteins result in neurodegenerative diseases. Mutations affecting microtubule motor function have been shown to cause hereditary forms of Charcot-Marie-Tooth disease (type 2A), hereditary spastic paraplegia and motor neuron disease. Although motor neurons appear to be uniquely susceptible to defects in axonal transport, recent work has identified links between perturbations in axonal transport and the pathogenesis of other neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. More broadly, cytoskeletal abnormalities might also be at the root of related disorders such as spinal muscular atrophy, supporting a key role for axonal transport in the pathogenesis of many neurodegenerative diseases.     

One-hit stochastic decline in a mechanochemical model of cytoskeleton-induced neuron death I: cell-fate arrival times

Much experimental evidence shows that the cytoskeleton is a downstream target and effector during cell death in numerous neurodegenerative diseases, including Parkinson's, Huntington's, and Alzheimer's diseases. However, recent evidence indicates that cytoskeletal dysfunction can also trigger neuronal death, by mechanisms as yet poorly understood. This is the first of two papers in which we study a mathematical model of cytoskeleton-induced neuron death. In our model, assembly control of the neuronal cytoskeleton interacts with both cellular stress levels and cytosolic free radical concentrations to trigger neurodegeneration. This trigger mechanism is further modulated by the presence of cell interactions in the form of a diffusible toxic factor released by dying neurons. We find that, consistent with empirical observations, our model produces one-hit exponential and sigmoid patterns of cell dropout. In all cases, cell dropout is exponential-tailed and described accurately by a gamma distribution. The transition between exponential and sigmoidal is gradual, and determined by a synergetic interaction between the magnitude of fluctuations in cytoskeleton assembly control and by the degree of cell coupling. We conclude that a single mechanism involving neuron interactions and fluctuations in cytoskeleton assembly control is compatible with the experimentally observed range of neuronal attrition kinetics.     

Amyloid fibrils of mammalian prion protein induce axonal degeneration in NTERA2-derived terminally differentiated neurons

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.     

Gigaxonin interacts with tubulin folding cofactor B and controls its degradation through the ubiquitin-proteasome pathway

Gigaxonin is mutated in human giant axonal neuropathy (GAN), an autosomal recessive neurodegenerative disorder. The presence of generalized cytoskeletal abnormalities , including few microtubules and accumulated intermediate filaments (IFs), in GAN suggests an essential role of gigaxonin in cytoskeletal organization and dynamics. However, the molecular mechanisms underlying the cytoskeletal pathology remain to be elucidated. Over the years, the ubiquitin-proteasome system (UPS) of intracellular protein degradation has been implicated in the control of many fundamental cellular processes. Defects in this system seem to be directly linked to the development of human diseases, including cancers and neurodegenerative diseases . Here, we show that gigaxonin controls protein degradation of tubulin folding cofactor B (TBCB) , a function disrupted by GAN-associated mutations. The substantial TBCB protein accumulation caused by impaired UPS may be a causative factor of cytoskeletal pathology in GAN. Our study provides important insight into pathogenesis of neurodegenerative diseases associated with cytoskeletal abnormalities.     

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.     

The synaptic cytoskeleton in development and disease

The cytoskeleton forms the backbone of neuronal architecture, sustaining its form and size, subcellular compartments and cargo logistics. The synaptic cytoskeleton can be categorized in the microtubule-based core cytoskeleton and the cortical membrane skeleton. While central microtubules form the fundamental basis for the construction of elaborate neuronal processes, including axons and synapses, cortical actin filaments are generally considered to function as mediators of synapse dynamics and plasticity. More recently, the submembranous network of spectrin and ankyrin molecules has been involved in the regulation of synaptic stability and maintenance. Disruption of the synaptic cytoskeleton primarily affects the stability and maturation of synapses but also secondarily disturbs neuronal communication. Consequently, a variety of inherited diseases are accompanied by cytoskeletal malfunctions, including spastic paraplegias, spinocerebellar ataxias, and mental retardation. Since the primary reasons for many of these diseases are still unknown model organisms with a conserved repertoire of cytoskeletal elements help to understand the underlying biological mechanisms. The astonishing technical as well as genetic accessibility of synapses in Drosophila has shown that loss of the cytoskeletal architecture leads to axonal transport defects, synaptic maturation deficits, and retraction of synaptic boutons, before synaptic terminals finally detach from their target cells, suggesting that similar processes could be involved in human neuronal diseases.     

Adrenomedullin,a Novel Target for Neurodegenerative Diseases

Neurodegenerative diseases represent a heterogeneous group of disorders whose common characteristic is the progressive degeneration of neuronal structure and function. Although much knowledge has been accumulated on the pathophysiology of neurodegenerative diseases over the years, more efforts are needed to understand the processes that underlie these diseases and hence to propose new treatments. Adrenomedullin (AM) is a multifunctional peptide involved in vasodilation, hormone secretion, antimicrobial defense, cellular growth, and angiogenesis. In neurons, AM and related peptides are associated with some structural and functional cytoskeletal proteins that interfere with microtubule dynamics. Furthermore, AM may intervene in neuronal dysfunction through other mechanisms such as immune and inflammatory response, apoptosis, or calcium dyshomeostasis. Alterations in AM expression have been described in neurodegenerative processes such as Alzheimer’s disease or vascular dementia. This review addresses the current state of knowledge on AM and its possible implication in neurodegenerative diseases.     

Neuronal polarization and the cytoskeleton

Neuronal polarization, the formation of one long axon and several short dendrites, is an obligatory process to integrate and propagate information within the brain. Axon formation is the key event during neuronal polarization and is based on tightly regulated rearrangements of the cytoskeleton. Here, we discuss how the cytoskeleton drives neuronal polarization. First, we convey the role of the actin cytoskeleton and microtubules during axon formation. Second, we discuss different cytoskeletal binding and regulating proteins, which are essential to specify the axon. Finally, we outline plus end tracking proteins (+TIPs) as important regulators for neuronal polarization by mediating the interaction between the actin cytoskeleton and microtubules and compare this function to other polarity processes.     

The parkinsonism producing neurotoxin MPP+ affects microtubule dynamics by acting as a destabilising factor

Dysfunction of the microtubule system is emerging as a contributing factor in a number of neurodegenerative diseases. Looking for the potential role played by the microtubule cytoskeleton in neuron degeneration underlying Parkinson's disease (PD), we investigate the influence of the parkinsonism producing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on microtubule dynamics. We find that it acts as a strong catastrophe promoter causing a decrease of the average length of microtubules assembled from purified tubulin. We also find that it reduces the number of microtubules nucleated from purified centrosomes. Finally, binding assays demonstrate that the neurotoxin binds specifically to tubulin in the microtubule lattice in a close to stoichiometric manner. This paper provides the first evidence that dynamic instability of microtubules is specifically affected by MPP+ and suggests that it could play a role in neuronal cell death underlying PD.     

Interleukin-1beta up-regulates expression of neurofilament light in human neuronal cells

Elevated expression of interleukin-1 (IL-1beta), a pro-inflammatory cytokine secreted by activated microglia, is a pathogenic marker of numerous neurodegenerative processes including Alzheimer's disease (AD). We have characterized a link between IL-1beta and the 68-kDa neurofilament light (NF-L) protein, which is a major component of the neuronal cytoskeleton. Using human brain aggregate cultures, we found that IL-1beta treatment significantly increased NF-L expression in primary neurons. Analysis of mRNA levels demonstrated elevated NF-L expression within 72 h while imaging of neurons by immunofluorescent staining for NF-L confirmed IL-1beta-induced NF-L protein expression. These observations suggest a potential inflammatory-induced mechanism for deregulation of an important cytoskeletal protein, NF-L, possibly leading to neuronal dysfunction.     

Computational predictions of volatile anesthetic interactions with the microtubule cytoskeleton: implications for side effects of general anesthesia

The cytoskeleton is essential to cell morphology, cargo trafficking, and cell division. As the neuronal cytoskeleton is extremely complex, it is no wonder that a startling number of neurodegenerative disorders (including but not limited to Alzheimer's disease, Parkinson's disease and Huntington's disease) share the common feature of a dysfunctional neuronal cytoskeleton. Recently, concern has been raised about a possible link between anesthesia, post-operative cognitive dysfunction, and the exacerbation of neurodegenerative disorders. Experimental investigations suggest that anesthetics bind to and affect cytoskeletal microtubules, and that anesthesia-related cognitive dysfunction involves microtubule instability, hyper-phosphorylation of the microtubule-associated protein tau, and tau separation from microtubules. However, exact mechanisms are yet to be identified. In this paper the interaction of anesthetics with the microtubule subunit protein tubulin is investigated using computer-modeling methods. Homology modeling, molecular dynamics simulations and surface geometry techniques were used to determine putative binding sites for volatile anesthetics on tubulin. This was followed by free energy based docking calculations for halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the tubulin body, and C-terminal regions for specific tubulin isotypes. Locations of the putative binding sites, halothane binding energies and the relation to cytoskeleton function are reported in this paper.     

Calcium signaling and molecular mechanisms underlying neurodegenerative diseases

Calcium (Ca²⁺) is a ubiquitous second messenger that regulates various activities in eukaryotic cells. Especially important role calcium plays in excitable cells. Neurons require extremely precise spatial-temporal control of calcium-dependent processes because they regulate such vital functions as synaptic plasticity. Recent evidence indicates that neuronal calcium signaling is abnormal in many of neurodegenerative disorders such as Alzheimer’s disease (AD), Huntington’s disease (HD) and Parkinson’s disease (PD). These diseases represent a major medical, social, financial and scientific problem, but despite enormous research efforts, they are still incurable and only symptomatic relief drugs are available. Thus, new approaches and targets are needed. This review highlight neuronal calcium-signaling abnormalities in these diseases, with particular emphasis on the role of neuronal store-operated Ca²⁺ entry (SOCE) pathway and its potential relevance as a therapeutic target for treatment of neurodegeneration.     

Neurodegenerative Diseases: Pathology and the Advantage of Single-Cell Profiling

The aggregation of neuronal proteins as inclusions is emerging as a common mechanistic theme in neurodegenerative diseases. The presence of these "disease-specific" pathologic changes in the brains of patients with neurodegenerative diseases assist pathologists in the diagnosis and characterization of dementing illnesses. However, these same inclusions may provide valuable clues toward understanding common pathologic roots and shared abnormalities in protein folding across disorders. Such an investigation will likely provide insights into disease mechanisms underlying neurodegenerative disorders characterized by abundant filamentous lesions. This review focuses on two themes: (i) Neurodegenerative disorders are characterized by shared and distinct histopathological and biochemical abnormalities, and (ii) the presence of abnormal protein aggregates may alter a gene, and hence protein expression in inclusion-bearing neurons predisposes them to dysfunction and eventual neuronal degeneration. The pathologic features of neurodegenerative diseases are first discussed followed by a rationale behind sampling mRNA species from single cells rather than from whole-brain homogenates to explore disease mechanisms.     

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.     

The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseases

Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca(2+)-dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A(2) (PLA(2)). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA(2), namely, the group IV cytosolic cPLA(2) and the group II secretory sPLA(2). A major focus is to elaborate the dual role of NADPH oxidase and PLA(2) in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA(2) will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases.     

Primary cilia in the postnatal brain: Subcellular compartments for organizing neuromodulatory signaling

Primary cilia have well characterized roles in early brain development, relaying signals critical for neurogenesis and brain formation during embryonic stages. Less understood are the contributions of cilia-mediated signaling to postnatal brain function. Several cilia-localized receptors that bind neuropeptides and neurotransmitters endogenous to the brain have been identified in adult neurons, but the functional significance of signaling through these cilia-localized receptors is largely unexplored. Ciliopathic disorders in humans often manifest with neurodevelopmental abnormalities and cognitive deficits. Intriguingly, recent research has also linked several neuropsychiatric disorders and neurodegenerative diseases to ciliary dysfunction. This review summarizes recent evidence suggesting that cilia signaling may dynamically regulate postnatal neuronal physiology and connectivity, and highlights possible links among cilia, neuronal circuitry, neuron survival, and neurological disorders.