ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear.

ALS-linked CCNF variant disrupts motor neuron ubiquitin homeostasis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that share pathological features, including the aberrant accumulation of ubiquitinated protein inclusions within motor neurons. Previously, we have shown that the sequestration of ubiquitin (Ub) into inclusions disrupts Ub homeostasis in cells expressing ALS-associated variants superoxide dismutase 1 (SOD1), fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). Here, we investigated whether an ALS/FTD-linked pathogenic variant in the CCNF gene, encoding the E3 Ub ligase Cyclin F (CCNF), also perturbs Ub homeostasis.

A Simple Differentiation Protocol for Generation of Induced Pluripotent Stem Cell-Derived Basal Forebrain-Like Cholinergic Neurons for Alzheimer's Disease and Frontotemporal Dementia Disease Modeling.

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors.

The mRNA-based reprogramming of fibroblasts from a SOD1 familial amyotrophic lateral sclerosis patient to induced pluripotent stem cell line UOWi007.

Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1 mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1 genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages.

Generation and characterization of a human induced pluripotent stem cell line UOWi005-A from dermal fibroblasts derived from a CCNF familial amyotrophic lateral sclerosis patient using mRNA reprogramming.

Dermal fibroblasts from a 59 year old male patient with amyotrophic lateral sclerosis (symptomatic at the time of collection), attributed to a mutation in the cyclin F gene (CCNF), were reprogrammed using mRNA and microRNA-delivered OSKM factors to induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed pluripotent, expressing typical pluripotency markers and were capable of three germ layer differentiation. This is the first reported reprogramming of cells with a mutation in the cyclin F gene, and represents a novel resource for the study of amyotrophic lateral sclerosis.

The Ubiquitin Proteasome System Is a Key Regulator of Pluripotent Stem Cell Survival and Motor Neuron Differentiation.

The ubiquitin proteasome system (UPS) plays an important role in regulating numerous cellular processes, and a dysfunctional UPS is thought to contribute to motor neuron disease. Consequently, we sought to map the changing ubiquitome in human iPSCs during their pluripotent stage and following differentiation to motor neurons. Ubiquitinomics analysis identified that spliceosomal and ribosomal proteins were more ubiquitylated in pluripotent stem cells, whilst proteins involved in fatty acid metabolism and the cytoskeleton were specifically ubiquitylated in the motor neurons.

Viral-free generation and characterization of a human induced pluripotent stem cell line from dermal fibroblasts.

Peripheral dermal fibroblasts (DF) from a healthy 56 year old female were obtained from the Centre for Healthy Brain Ageing (CHeBA) Biobank, University of New South Wales, under the material transfer agreement with the University of Wollongong. DFs were reprogrammed via mRNA-delivered transcription factors into induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed to be pluripotent, capable of three germ layer differentiation and are thus a useful resource for creating iPSC-derived healthy human cells of any lineage.

Generation and characterization of human induced pluripotent stem cell lines from a familial Alzheimer's disease PSEN1 A246E patient and a non-demented family member bearing wild-type PSEN1.

The induced pluripotent stem cell (iPSC) lines UOWi002-A and UOWi003-A were reprogrammed from dermal fibroblasts via mRNA transfection. Dermal fibroblasts from a 56 year old female caucasian familial Alzheimer's disease patient carrying A246E mutation in the PSEN1 gene (familial AD3, autopsy confirmed Alzheimer's disease) and a 75 year old female non-demented control from the same family bearing the wild-type PSEN1 A246 genotype were obtained from the Coriell Institute (AG06848 and AG06846, respectively). The generated iPSCs were characterized and pluripotency was confirmed.

Impairments in Motor Neurons, Interneurons and Astrocytes Contribute to Hyperexcitability in ALS: Underlying Mechanisms and Paths to Therapy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of motor neurons leading to progressive paralysis and death. Using transcranial magnetic stimulation (TMS) and nerve excitability tests, several clinical studies have identified that cortical and peripheral hyperexcitability are among the earliest pathologies observed in ALS patients. The changes in the electrophysiological properties of motor neurons have been identified in both sporadic and familial ALS patients, despite the diverse etiology of the disease.

Common pitfalls of stem cell differentiation: a guide to improving protocols for neurodegenerative disease models and research.

Induced pluripotent stem cells and embryonic stem cells have revolutionized cellular neuroscience, providing the opportunity to model neurological diseases and test potential therapeutics in a pre-clinical setting. The power of these models has been widely discussed, but the potential pitfalls of stem cell differentiation in this research are less well described. We have analyzed the literature that describes differentiation of human pluripotent stem cells into three neural cell types that are commonly used to study diseases, including forebrain cholinergic neurons for Alzheimer's disease, midbrain dopaminergic neurons for Parkinson's disease and cortical astrocytes for neurodegenerative and psychiatric disorders.

SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation.

Background: Amyotrophic Lateral Sclerosis is characterized by a focal onset of symptoms followed by a progressive spread of pathology that has been likened to transmission of infectious prions. Cell-to-cell transmission of SOD1 protein aggregates is dependent on fluid-phase endocytosis pathways, although the precise molecular mechanisms remain to be elucidated.

Results: We demonstrate in this paper that SOD1 aggregates interact with the cell surface triggering activation of Rac1 and subsequent membrane ruffling permitting aggregate uptake via stimulated macropinocytosis.