Lipid levels correlate with neuronal and dopaminergic markers during the differentiation of SH-SY5Y cells.

Parkinson's Disease (PD) is characterised by the loss of dopaminergic neurons and the deposition of protein inclusions called Lewy Bodies (LBs). LBs are heterogeneous structures composed of protein and lipid molecules and their main constituent is the presynaptic protein α-synuclein. SH-SY5Y cells are neuroblastoma cells commonly used to model PD because they express dopaminergic markers and α-synuclein and they can be differentiated into neuronal cells using established protocols. Despite increasing evidence pointing towards a role of lipids in PD, limited knowledge is available on the lipidome of undifferentiated and differentiated SH-SY5Y cells. Using a combination of lipidomics, proteomics, morphological and electrophysiological measurements, we identified specific lipids, including sphingolipids, whose levels are affected by the differentiation of SH-SY5Y neuroblastoma cells and found that the levels of these lipids correlate with those of neuronal and dopaminergic markers. These results provide a quantitative characterisation of the changes in lipidome associated with the differentiation of SH-SY5Y cells into more neuronal and dopaminergic-like phenotype and serve as a basis for further characterisation of lipid disruptions in association with PD and its risk factors in this dopaminergic-like neuronal cell model.

Unbiased Label-Free Quantitative Proteomics of Cells Expressing Amyotrophic Lateral Sclerosis (ALS) Mutations in CCNF Reveals Activation of the Apoptosis Pathway: A Workflow to Screen Pathogenic Gene Mutations.

The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a diverse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1/p62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an example, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of in vitro and in vivo models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein-protein and protein-substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies.

The interplay between Glucocerebrosidase, α-synuclein and lipids in human models of Parkinson's disease.

Mutations in the gene GBA, encoding glucocerebrosidase (GCase), are the highest genetic risk factor for Parkinson's disease (PD). GCase is a lysosomal glycoprotein responsible for the hydrolysis of glucosylceramide into glucose and ceramide. Mutations in GBA cause a decrease in GCase activity, stability and protein levels which in turn lead to the accumulation of GCase lipid substrates as well as α-synuclein (αS) in vitro and in vivo. αS is the main constituent of Lewy bodies found in the brain of PD patients and an increase in its levels was found to be associated with a decrease in GCase activity/protein levels in vitro and in vivo. In this review, we describe the reported biophysical and biochemical changes that GBA mutations can induce in GCase activity and stability as well as the current overview of the levels of GCase protein/activity, αS and lipids measured in patient-derived samples including post-mortem brains, stem cell-derived neurons, cerebrospinal fluid, blood and fibroblasts as well as in SH-SY5Y cells. In particular, we report how the levels of αS and lipids are affected by/correlated to significant changes in GCase activity/protein levels and which cellular pathways are activated or disrupted by these changes in each model. Finally, we review the current strategies used to revert the changes in the levels of GCase activity/protein, αS and lipids in the context of PD.

Generation of APOE knock-down SK-N-SH human neuroblastoma cells using CRISPR/Cas9: a novel cellular model relevant to Alzheimer's disease research.

APOE ε4 is the major genetic risk factor for Alzheimer's disease (AD). A precise role for apolipoprotein E (apoE) in the pathogenesis of the disease remains unclear in part due to its expression in multiple cell types of the brain. APOE is highly expressed in astrocytes and microglia, however its expression can also be induced in neurons under various conditions. The neuron-like cell line SK-N-SH is a useful model in the study of the cellular and molecular effects of apoE as it can be differentiated with retinoic acid to express and secrete high levels of apoE and it also shows the same apoE fragmentation patterns observed in the human brain. We previously found that apoE is cleaved into a 25-kDa fragment by high temperature-requirement serine protease A1 (HtrA1) in SK-N-SH cells. To further understand the endogenous functions of apoE, we used CRISPR/Cas9 to generate SK-N-SH cell lines with APOE expression knocked-down (KD). APOE KD cells showed lower APOE and HTRA1 expression than parental SK-N-SH cells but no overt differences in neuritogenesis or cell proliferation compared with the CRISPR/Cas9 control cells. This research shows that the loss of apoE and HtrA1 has a negligible effect on neuritogenesis and cell survival in SK-N-SH neuron-like cells.

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. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.

The mRNA-based reprogramming of fibroblasts from a SOD1E101G 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 SOD1E101G 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 SOD1E101G genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1E101G mutation.

Generation and characterization of a human induced pluripotent stem cell line UOWi005-A from dermal fibroblasts derived from a CCNFS621G 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 (CCNFS621G), 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.

Understanding the Role of ApoE Fragments in Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most devastating neurodegenerative diseases. It has been known for decades that the APOE ɛ4 allele is the most significant genetic risk factor for late-onset AD and yet its precise role in the disease remains unclear. The APOE gene encodes apolipoprotein E (apoE), a 35 kDa glycoprotein highly expressed in the brain. There are three different isoforms: apoE3 is the most common allele in the population, whilst apoE2 decreases, and apoE4 increases AD risk. ApoE has numerous functions that affect neuronal and non-neuronal cells, thus how it contributes to disease onset and progression is hotly debated. The apoE4 isoform has been linked to the accumulation of both of the major pathological hallmarks of AD, amyloid plaques containing amyloid ß peptides, and neurofibrillary tangles containing hyperphosphorylated tau protein, as well as other hallmarks of the disease, including inflammation and oxidative stress. Numerous studies have shown that apoE undergoes fragmentation in the human brain, and that the fragmentation pattern varies between isoforms. It was previously shown that apoE4 has neurotoxic functions, however recent data has also identified a neuroprotective role for the apoE N-terminal 25 kDa fragment, which is more prevalent in apoE3 individuals. The ability of the apoE 25 kDa fragment to promote neurite outgrowth was recently demonstrated and this suggests there is a potential loss of neuroprotection in apoE4 individuals in addition to the previously described gain of toxic function for specific apoE4 fragments. Here we review the enzymes proposed to be responsible for apoE fragmentation, the specific functions of different apoE fragments and their possible links with AD.

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. Resource table.

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. The PSEN1 genotype was maintained in both iPSC lines. Resource table.

The serine protease HtrA1 contributes to the formation of an extracellular 25-kDa apolipoprotein E fragment that stimulates neuritogenesis.

Apolipoprotein-E (apoE) is a glycoprotein highly expressed in the brain, where it appears to play a role in lipid transport, ß-amyloid clearance, and neuronal signaling. ApoE proteolytic fragments are also present in the brain, but the enzymes responsible for apoE fragmentation are unknown, and the biological activity of specific apoE fragments remains to be determined. Here we utilized SK-N-SH neuroblastoma cells differentiated into neurons with all-trans-retinoic acid (ATRA) to study extracellular apoE proteolysis. ApoE fragments were detectable in culture supernatants after 3 days, and their levels were increased for up to 9 days in the presence of ATRA. The concentration of apoE fragments was positively correlated with levels of the neuronal maturation markers (PSD95 and SMI32). The most abundant apoE fragments were 25- and 28-kDa N-terminal fragments that both contained sialylated glycosylation and bound to heparin. We detected apoE fragments only in the extracellular milieu and not in cell lysates, suggesting that an extracellular protease contributes to apoE fragmentation. Of note, siRNA-mediated knockdown of high-temperature requirement serine peptidase A1 (HtrA1) and a specific HtrA1 inhibitor reduced apoE 25-kDa fragment formation by 41 and 86%, respectively. Recombinant 25-kDa fragment apoE and full-length apoE both stimulated neuritogenesis in vitro, increasing neuroblastoma neurite growth by more than 2-fold over a 6-day period. This study provides a cellular model for assessing apoE proteolysis, indicates that HtrA1 regulates apoE 25-kDa fragment formation under physiological conditions, and reveals a new neurotrophic function for the apoE 25-kDa fragment.

Neuroprotective effects of apigenin against inflammation, neuronal excitability and apoptosis in an induced pluripotent stem cell model of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases, yet current therapeutic treatments are inadequate due to a complex disease pathogenesis. The plant polyphenol apigenin has been shown to have anti-inflammatory and neuroprotective properties in a number of cell and animal models; however a comprehensive assessment has not been performed in a human model of AD. Here we have used a human induced pluripotent stem cell (iPSC) model of familial and sporadic AD, in addition to healthy controls, to assess the neuroprotective activity of apigenin. The iPSC-derived AD neurons demonstrated a hyper-excitable calcium signalling phenotype, elevated levels of nitrite, increased cytotoxicity and apoptosis, reduced neurite length and increased susceptibility to inflammatory stress challenge from activated murine microglia, in comparison to control neurons. We identified that apigenin has potent anti-inflammatory properties with the ability to protect neurites and cell viability by promoting a global down-regulation of cytokine and nitric oxide (NO) release in inflammatory cells. In addition, we show that apigenin is able to protect iPSC-derived AD neurons via multiple means by reducing the frequency of spontaneous Ca(2+) signals and significantly reducing caspase-3/7 mediated apoptosis. These data demonstrate the broad neuroprotective action of apigenin against AD pathogenesis in a human disease model.

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. Published protocols for differentiation vary widely in the reported efficiency of target cell generation. Additionally, characterization of the cells by expression profile and functionality differs between studies and is often insufficient, leading to highly variable protocol outcomes. We have synthesized this information into a simple methodology that can be followed when performing or assessing differentiation techniques. Finally we propose three considerations for future research, including the use of physiological O2 conditions, three-dimensional co-culture systems and microfluidics to control feeding cycles and growth factor gradients. Following these guidelines will help researchers to ensure that robust and meaningful data is generated, enabling the full potential of stem cell differentiation for disease modeling and regenerative medicine.

Apolipoprotein D modulates amyloid pathology in APP/PS1 Alzheimer's disease mice.

Apolipoprotein D (apoD) is expressed in the brain and levels are increased in affected brain regions in Alzheimer's disease (AD). The role that apoD may play in regulating AD pathology has not been addressed. Here, we crossed both apoD-null mice and Thy-1 human apoD transgenic mice with APP-PS1 amyloidogenic AD mice. Loss of apoD resulted in a nearly 2-fold increase in hippocampal amyloid plaque load, as assessed by immunohistochemical staining. Conversely, transgenic expression of neuronal apoD reduced hippocampal plaque load by approximately 35%. This latter finding was associated with a 60% decrease in amyloid ß 1-40 peptide levels, and a 34% decrease in insoluble amyloid ß 1-42 peptide. Assessment of ß-site amyloid precursor protein cleaving enzyme-1 (BACE1) levels and proteolytic products of amyloid precursor protein and neuregulin-1 point toward a possible association of altered BACE1 activity in association with altered apoD levels. In conclusion, the current studies provide clear evidence that apoD regulates amyloid plaque pathology in a mouse model of AD.

Altered ceramide acyl chain length and ceramide synthase gene expression in Parkinson's disease.

Genetic studies have provided increasing evidence that ceramide homeostasis plays a role in neurodegenerative diseases including Parkinson's disease (PD). It is known that the relative amounts of different ceramide molecular species, as defined by their fatty acyl chain length, regulate ceramide function in lipid membranes and in signaling pathways. In the present study we used a comprehensive sphingolipidomic case-control approach to determine the effects of PD on ceramide composition in postmortem brain tissue from the anterior cingulate cortex (a region with significant PD pathology) and the occipital cortex (spared in PD), also assessing mRNA expression of the major ceramide synthase genes that regulate ceramide acyl chain composition in the same tissue using quantitative PCR. In PD anterior cingulate cortex but not occipital cortex, total ceramide and sphingomyelin levels were reduced from control levels by 53% (P < 0.001) and 42% (P < 0.001), respectively. Of the 13 ceramide and 15 sphingomyelin molecular lipid species identified and quantified, there was a significant shift in the ceramide acyl chain composition toward shorter acyl chain length in the PD anterior cingulate cortex. This PD-associated change in ceramide acyl chain composition was accompanied by an upregulation of ceramide synthase-1 gene expression, which we consider may represent a response to reduced ceramide levels. These data suggest a significant shift in ceramide function in lipid membranes and signaling pathways occurs in regions with PD pathology. Identifying the regulatory mechanisms precipitating this change may provide novel targets for future therapeutics.