Search Results (14)

Impairment of axonal transport may contribute to the degeneration of dopaminergic (DAergic) neurons in the substantia nigra (SN), a key event in Parkinson’s disease (PD) pathogenesis. Due to the lack of early diagnosis, changes in axonal transport at the preclinical stage can only be studied in PD models. We assessed gene expression (RT-PCR after cell sorting) and protein levels (semiquantitative immunohistochemistry) of axonal transport-related proteins in SN DAergic neurons from mice in subchronic MPTP models of PD (preclinical and clinical stages) and controls. The proteins studied included α-tubulin (Tuba1a), β-tubulin (Tubb3), kinesin (Kif5b, Klc1), dynein (Dynll1, Dync1i1), dynactin (Dctn1), microtubule affinity-regulating kinase 1 (Mark1), and tau (Mapt). In the preclinical stage, Kif5b expression and Kif5B level were increased, possibly to compensatorily preserve anterograde transport. Dynll1 and Tuba1a were upregulated, whereas Dync1i1 and Mapt were downregulated, with no change in tubulin or tau protein levels. In the clinical stage, Klc1, Dync1i1, Dctn1, Mark1, and Mapt expression and Kif5B protein levels decreased. These data indicate that transcriptional alterations in axonal transport proteins precede protein-level changes in DAergic neurons. The upregulation of Kif5B in the preclinical stage suggests that axonal transport proteins may serve as potential early therapeutic targets in PD. Full article

Mutations in the gene encoding the p150 subunit of the dynactin complex (DCTN1) are linked to amyotrophic lateral sclerosis, spinal and bulbar muscular atrophy, and Perry syndrome. These neurodegenerative diseases can cause muscle weakness and atrophy, parkinsonian-like symptoms, and paralysis. To examine the evolution of neuropathology caused by a mutation in DCTN1 and cellular mechanisms of disease for therapeutic discovery, we characterized mice expressing either human wildtype or mutant (G59S) DCTN1. Neuron-specific expression of mutant, but not wildtype, DCTN1 caused fatal age-related paralytic disease and motor neuron (MN) degeneration in the spinal cord with axonopathy and chromatolysis without apoptotic morphology. MNs became positive for cleaved caspase-3, cleaved caspase-8, and nitrated Hsp90. Mitochondria accumulated and appeared fragmented and dysmorphic and then were lost. This pathology was accompanied by invasion of CD95- and CD8-positive mononuclear T cells into the ventral horn and accumulation of TNFα and IL9. Administration of the mitochondrial division inhibitor-1 (Mdivi-1) protected MNs and extended the lifespan of G59S-DCTN1 mice. A mitochondrial permeability transition pore inhibitor also extended lifespan. Thus, mutant DCTN1 causes degeneration of MNs associated with axonopathy, mitochondriopathy, nitrative stress, and caspase activation. It appears as retrograde neurodegeneration and inflammatory T-cell-like cytolysis. Mitochondria are possible therapeutic targets in DCTN1-linked neurodegenerative disorders. Full article

Dynein is a minus-end-directed microtubule motor that transports a variety of cargoes. Cargo specificity is mediated by a class of adaptor proteins that bind to the interface between dynein and dynactin, along the length of the Arp1 filament of dynactin, and that co-activate the motor. NuMA, ninein, and ninein-like protein (Nlp) are cargo-adaptors that are involved in microtubule organization, rather than carrying portable cargoes. At the same time, ninein and Nlp are believed to be anchorage factors for gamma-tubulin ring complexes to the centrosome. Here, we discuss recent findings on the interaction of NuMA and ninein with the dynein/dynactin complex, and how these findings challenge earlier concepts on ninein-dependent microtubule organization via gamma-tubulin complexes. We do not intend to provide an encyclopedic review on NuMA and ninein, but rather develop a hypothesis about how conformational changes may regulate the activities and binding specificities of these two proteins. Full article

Background/Objectives: Proper regulation of copper is essential for maintaining neuronal stability and is facilitated by several chaperone proteins, protecting cells from oxidative damage that would otherwise be caused by improperly regulated copper ions. Oxidative stress, resulting from such dysregulation, is hypothesized to play a significant role in the pathogenesis of Alzheimer’s disease (AD). Methods: In this study, we evaluated the concentrations of the copper chaperones CCS, DCTN4, and ATOX1 in control and AD cases via Western blotting and ELISA, and quantified the copper concentrations in fractionated neurons using ICP-MS. Results: Our findings reveal a significant reduction in CCS levels in AD cases (p = 0.0085), with a progressive decline observed with advancing age. This decline was more pronounced in women, although the difference did not reach statistical significance (p = 0.0768). No significant differences were observed in copper concentrations within synaptosomal (p = 0.3869) or cytosolic fractions (p = 0.4461) between the AD and control cases. Additionally, comprehensive analyses of the effects of sex and age showed no significant impact on the levels of copper chaperones or copper distribution across cellular compartments. Conclusions: These results suggest a strong association between reduced CCS levels and AD pathology, highlighting a potential role for CCS in the redistribution of copper ions within neurons. This redistribution may contribute to oxidative stress and neuronal dysfunction, offering new insights into the mechanisms underlying AD pathogenesis. Full article

Multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) are uncommon multifactorial atypical Parkinsonian syndromes, expressed by various clinical features. MSA and PSP are commonly considered sporadic neurodegenerative disorders; however, our understanding is improving of their genetic framework. The purpose of this study was to critically review the genetics of MSA and PSP and their involvement in the pathogenesis. A systemized literature search of PubMed and MEDLINE was performed up to 1 January 2023. Narrative synthesis of the results was undertaken. In total, 43 studies were analyzed. Although familial MSA cases have been reported, the hereditary nature could not be demonstrated. COQ2 mutations were involved in familial and sporadic MSA, without being reproduced in various clinical populations. In terms of the genetics of the cohort, synuclein alpha (SNCA) polymorphisms were correlated with an elevated likelihood of manifesting MSA in Caucasians, but a causal effect relationship could not be demonstrated. Fifteen MAPT mutations were linked with PSP. Leucine-rich repeat kinase 2 (LRRK2) is an infrequent monogenic mutation of PSP. Dynactin subunit 1 (DCTN1) mutations may imitate the PSP phenotype. GWAS have noted many risk loci of PSP (STX6 and EIF2AK3), suggesting pathogenetic mechanisms related to PSP. Despite the limited evidence, it seems that genetics influence the susceptibility to MSA and PSP. MAPT mutations result in the MSA and PSP pathologies. Further studies are crucial to elucidate the pathogeneses of MSA and PSP, which will support efforts to develop novel drug options. Full article

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1^G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies. Full article

Dementia has an enormous impact on medical and financial resources in aging societies like Japan. Diagnosis of dementia can be made by physical and mental examinations, imaging tests, and findings of high abnormal proteins in cerebrospinal fluids. In addition, genetic tests can be performed in neurodegenerative diseases such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), and Parkinson’s disease (PD). In this case series, we presented three cases of dementia with unknown causes who carry novel variants in the genes associated with neurodegenerative diseases. Three patients (Patients 1, 2, and 6) were found by screening 18 dementia patients using a gene panel including 63 genes. The age of onset for Patient 1 was 74 years old, and his father had PD and mother had AD. The age of onset for Patient 2 was 75 years old, and her mother had AD. The age of onset for Patient 6 was 83 years old, and her father, two sisters, and daughter had dementia. The Mini-Mental State Examination produced results of 20, 15, and 22, respectively. The suspected diagnosis by neurological examinations and imaging studies for Patients 1 and 2 was AD, and for Patient 6 was FTD. Patient 1 was treated with donepezil; Patient 2 was treated with donepezil and memantine; and Patient 6 was treated with donepezil, galantamine, and rivastigmine. The three rare variants identified were: CLCN1, encoding a chloride channel, c.2848G>A:p.Glu950Lys (Patient 1); RYR2, encoding a calcium releasing ryanodine receptor, c.13175A>G:p.Lys4392Arg (Patient 2); and DCTN1, encoding a subunit of dynactin, c. 3209G>A:p.Arg1070Gln (Patient 6). The detected variants were interpreted according to the American College of Medical Genetics (ACMG) guidelines. The minor allele frequency for each variant was 0.025%, 0.023%, and 0.0004% in East Asians, respectively. The DCTN1 variant found in Patient 6 might be associated with FTD. Although none of them were previously reported in dementia patients, all variants were classified as variants of unknown significance (VUS). Our report suggests that results of genetic tests in elderly patients with dementia need to be carefully interpreted. Further data accumulation of genotype–phenotype relationships and development of appropriate functional models are warranted. Full article

Understanding methylation dynamics in organs or tissues containing many different cell types is a challenging task that cannot be efficiently addressed by the low-depth bisulphite sequencing of DNA extracted from such sources. Here we explored the feasibility of ultra-deep bisulphite sequencing of long amplicons to reveal the brain methylation patterns in three selected honey bee genes analysed across five distinct conditions on the Illumina MiSeq platform. By combing 15 libraries in one run we achieved a very high sequencing depth of 240,000–340,000 reads per amplicon, suggesting that most of the cell types in the honey bee brain, containing approximately 1 million neurons, are represented in this dataset. We found a small number of gene-specific patterns for each condition in individuals of different ages and performing distinct tasks with 80–90% of those were represented by no more than a dozen patterns. One possibility is that such a small number of frequent patterns is the result of differentially methylated epialleles, whereas the rare and less frequent patterns reflect activity-dependent modifications. The condition-specific methylation differences within each gene appear to be position-dependent with some CpGs showing significant changes and others remaining stable in a methylated or non-methylated state. Interestingly, no significant loss of methylation was detected in very old individuals. Our findings imply that these diverse patterns represent a special challenge in the analyses of DNA methylation in complex tissues and organs that cannot be investigated by low-depth genome-wide bisulphite sequencing. We conclude that ultra-deep sequencing of gene-specific amplicons combined with genotyping of differentially methylated epialleles is an effective way to facilitate more advanced neuro-epigenomic studies in honey bees and other insects. Full article

A novel cosegregating splice site variant in the Dynactin-1 (DCTN1) gene was discovered by Next Generation Sequencing (NGS) in a family with a history of bipolar disorder (BD) and major depressive diagnosis (MDD). Psychiatric illness in this family follows an autosomal dominant pattern. DCTN1 codes for the largest dynactin subunit, namely p150^Glued, which plays an essential role in retrograde axonal transport and in neuronal autophagy. A GT→TT transversion in the DCTN1 gene, uncovered in the present work, is predicted to disrupt the invariant canonical splice donor site IVS22 + 1G > T and result in intron retention and a premature termination codon (PTC). Thus, this splice site variant is predicted to trigger RNA nonsense-mediated decay (NMD) and/or result in a C-terminal truncated p150^Glued protein (ct-p150^Glued), thereby negatively impacting retrograde axonal transport and neuronal autophagy. BD prophylactic medications, and most antipsychotics and antidepressants, are known to enhance neuronal autophagy. This variant is analogous to the dominant-negative GLUED Gl¹ mutation in Drosophila, which is responsible for a neurodegenerative phenotype. The newly identified variant may reflect an autosomal dominant cause of psychiatric pathology in this affected family. Factors that affect alternative splicing of the DCTN1 gene, leading to NMD and/or ct-p150^Glued, may be of fundamental importance in contributing to our understanding of the etiology of BD as well as MDD. Full article

Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here, we present loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. We investigated the requirements of Spindly protein domains for its localisation and function, and found that the carboxy-terminal region controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila. Full article

African swine fever virus (ASFV) is a double-stranded DNA virus causing a hemorrhagic fever disease with high mortality rates and severe economic losses in pigs worldwide. ASFV replicates in perinuclear sites called viral factories (VFs) that are morphologically similar to cellular aggresomes. This fact raises the possibility that both VFs and aggresomes may be the same structure. However, little is known about the process involved in the formation of these viral replication platforms. In order to expand our knowledge on the assembly of ASFV replication sites, we have analyzed the involvement of both canonical aggresome pathways in the formation of ASFV VFs: HDAC6 and BAG3. HDAC6 interacts with a component of the dynein motor complex (dynactin/p150^Glued) and ubiquitinated proteins, transporting them to the microtubule-organizing center (MTOC) and leading to aggresome formation, while BAG3 is mediating the recruitment of non-ubiquitinated proteins through a similar mechanism. Tubacin-mediated HDAC6 inhibition and silencing of BAG3 pathways, individually or simultaneously, did not prevent ASFV VF formation. These findings show that HDAC6 and Bag3 are not required for VFs formation suggesting that aggresomes and VFs are not the same structures. However, alternative unexplored pathways may be involved in the formation of aggresomes. Full article

Extracellular-signal regulated kinase (ERK) signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2) as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150^Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150^Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin. Full article

Motor neurons typically have very long axons, and fine-tuning axonal transport is crucial for their survival. The obstruction of axonal transport is gaining attention as a cause of neuronal dysfunction in a variety of neurodegenerative motor neuron diseases. Depletions in dynein and dynactin-1, motor molecules regulating axonal trafficking, disrupt axonal transport in flies, and mutations in their genes cause motor neuron degeneration in humans and rodents. Axonal transport defects are among the early molecular events leading to neurodegeneration in mouse models of amyotrophic lateral sclerosis (ALS). Gene expression profiles indicate that dynactin-1 mRNA is downregulated in degenerating spinal motor neurons of autopsied patients with sporadic ALS. Dynactin-1 mRNA is also reduced in the affected neurons of a mouse model of spinal and bulbar muscular atrophy, a motor neuron disease caused by triplet CAG repeat expansion in the gene encoding the androgen receptor. Pathogenic androgen receptor proteins also inhibit kinesin-1 microtubule-binding activity and disrupt anterograde axonal transport by activating c-Jun N-terminal kinase. Disruption of axonal transport also underlies the pathogenesis of spinal muscular atrophy and hereditary spastic paraplegias. These observations suggest that the impairment of axonal transport is a key event in the pathological processes of motor neuron degeneration and an important target of therapy development for motor neuron diseases. Full article

Unlike transport vesicles or organelles, human adenovirus (HAdV) directly binds to the microtubule minus end-directed motor dynein for transport to the nucleus. The dynein cofactor dynactin enhances nuclear transport of HAdV and boosts infection. To determine if dynactin has a specific role in cytoplasmic trafficking of incoming HAdV on microtubules, we used live cell spinning disc confocal microscopy at 25 Hz acquisition frequency and automated tracking of single virus particles at 20–50 nm spatial resolution. Computational dissection by machine-learning algorithms extracted specific motion patterns of viral trajectories. We found that unperturbed cells supported two kinds of microtubule-dependent motions, directed motions (DM) and fast drifts (FD). DM had speeds of 0.2 to 2 µm/s and run lengths of 0.4 up to 7 µm, while FD were slower and less extensive at 0.02 to 0.4 µm/s and 0.05 to 2.5 µm. Dynactin interference by overexpression of p50/dynamitin or a coiled-coil domain of p150/Glued reduced the speeds and amounts of both center- and periphery-directed DM but not FD, and inhibited infection. These results indicate that dynactin enhances adenovirus infection by increasing the speed and efficiency of dynein-mediated virus motion to the nucleus, and, surprisingly, also supports a hereto unknown motor activity for virus transport to the cell periphery. Full article