Distinct transcriptional alterations distinguish Lewy body disease from Alzheimer's disease.

Lewy body dementia and Alzheimer's disease (AD) are leading causes of cognitive impairment, characterized by distinct but overlapping neuropathological hallmarks. Lewy body disease (LBD) is characterized by alpha-synuclein aggregates in the form of Lewy bodies as well as the deposition of extracellular amyloid plaques, with many cases also exhibiting neurofibrillary tangle (NFT) pathology. In contrast, Alzheimer's disease is characterized by amyloid plaques and neurofibrillary tangles. Both conditions often co-occur with additional neuropathological changes, such as vascular disease and TDP-43 pathology. To elucidate shared and distinct molecular signatures underlying these mixed neuropathologies, we extensively analyzed transcriptional changes in the anterior cingulate cortex, a brain region critically involved in cognitive processes. We performed bulk tissue RNAseq from the anterior cingulate cortex and determined differentially expressed genes (q-value < 0.05) in control (n = 81), Lewy body disease (n = 436), Alzheimer's disease (n = 53), and pathological amyloid cases consisting of amyloid pathology with minimal or no tau pathology (n = 39). We used gene set enrichment and weighted gene correlation network analysis (WGCNA) to understand the pathways associated with each neuropathologically defined group. Lewy body disease cases had strong up-regulation of inflammatory pathways and down-regulation of metabolic pathways. The Lewy body disease cases were further subdivided into either high Thal amyloid, Braak NFT, or low pathological burden cohorts. Compared to the control cases, the Lewy body disease cohorts consistently showed up-regulation for genes involved in protein folding and cytokine immune response, as well as down-regulation of fatty acid metabolism. Surprisingly, concomitant tau pathology within the Lewy body disease cases resulted in no additional changes. Some core inflammatory pathways were shared between Alzheimer's disease and Lewy body disease but with numerous disease-specific changes. Direct comparison of Lewy body disease cohorts versus Alzheimer's disease cases revealed strong enrichment of synaptic signaling, behavior, and neuronal system pathways. Females had a stronger response overall in both Lewy body and Alzheimer's disease, with several sex-specific changes. Overall, the results identify genes commonly and uniquely dysregulated in neuropathologically defined Lewy body disease and Alzheimer's disease cases, shedding light on shared and distinct molecular pathways. Additionally, the study underscores the importance of considering sex-specific changes in understanding the complex transcriptional landscape of these neurodegenerative diseases.

Widespread choroid plexus contamination in sampling and profiling of brain tissue.

The choroid plexus, a tissue responsible for producing cerebrospinal fluid, is found predominantly in the lateral and fourth ventricles of the brain. This highly vascularized and ciliated tissue is made up of specialized epithelial cells and capillary networks surrounded by connective tissue. Given the complex structure of the choroid plexus, this can potentially result in contamination during routine tissue dissection. Bulk and single-cell RNA sequencing studies, as well as genome-wide in situ hybridization experiments (Allen Brain Atlas), have identified several canonical markers of choroid plexus such as Ttr, Folr1, and Prlr. We used the Ttr gene as a marker to query the Gene Expression Omnibus database for transcriptome studies of brain tissue and identified at least some level of likely choroid contamination in numerous studies that could have potentially confounded data analysis and interpretation. We also analyzed transcriptomic datasets from human samples from Allen Brain Atlas and the Genotype-Tissue Expression (GTEx) database and found abundant choroid contamination, with regions in closer proximity to choroid more likely to be impacted such as hippocampus, cervical spinal cord, substantia nigra, hypothalamus, and amygdala. In addition, analysis of both the Allen Brain Atlas and GTEx datasets for differentially expressed genes between likely "high contamination" and "low contamination" groups revealed a clear enrichment of choroid plexus marker genes and gene ontology pathways characteristic of these ciliated choroid cells. Inclusion of these contaminated samples could result in biological misinterpretation or simply add to the statistical noise and mask true effects. We cannot assert that Ttr or other genes/proteins queried in targeted assays are artifacts from choroid contamination as some of these differentials may be due to true biological effects. However, for studies that have an unequal distribution of choroid contamination among groups, investigators may wish to remove contaminated samples from analyses or incorporate choroid marker gene expression into their statistical modeling. In addition, we suggest that a simple RT-qPCR or western blot for choroid markers would mitigate unintended choroid contamination for any experiment, but particularly for samples intended for more costly omic profiling. This study highlights an unexpected problem for neuroscientists, but it is also quite possible that unintended contamination of adjacent structures occurs during dissections for other tissues but has not been widely recognized.

The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease.

Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.

The Hitchhiker's Guide to Nucleocytoplasmic Trafficking in Neurodegeneration.

The widespread nature of nucleocytoplasmic trafficking defects and protein accumulation suggests distinct yet overlapping mechanisms in a variety of neurodegenerative diseases. Detailed understanding of the cellular pathways involved in nucleocytoplasmic transport and its dysregulation are essential for elucidating neurodegenerative pathogenesis and pinpointing potential areas for therapeutic intervention. The transport of cargos from the nucleus to the cytoplasm is generally regulated by the structure and function of the nuclear pore as well as the karyopherin α/ß, importin, exportin, and mRNA export mechanisms. The disruption of these crucial transport mechanisms has been extensively described in the context of neurodegenerative diseases. One common theme in neurodegeneration is the cytoplasmic aggregation of proteins, including nuclear RNA binding proteins, repeat expansion associated gene products, and tau. These cytoplasmic aggregations are partly a consequence of failed nucleocytoplasmic transport machinery, but can also further disrupt transport, creating cyclical feed-forward mechanisms that exacerbate neurodegeneration. Here we describe the canonical mechanisms that regulate nucleocytoplasmic trafficking as well as how these mechanisms falter in neurodegenerative diseases.

ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.

Correction to: ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The original article was published erroneously without mentioning the support of the U.S.

Moderate intrinsic phenotypic alterations in C9orf72 ALS/FTD iPSC-microglia despite the presence of C9orf72 pathological features.

While motor and cortical neurons are affected in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), it remains largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We differentiated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels, and generate dipeptide repeat proteins. Healthy control and C9orf72 ALS/FTD iPSC-MG equally express microglial specific genes and perform microglial functions, including inflammatory cytokine release and phagocytosis of extracellular cargos, such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. RNA sequencing analysis revealed select transcriptional changes of genes associated with neuroinflammation or neurodegeneration in diseased microglia yet no significant differentially expressed microglial-enriched genes. Moderate molecular and functional differences were observed in C9orf72 iPSC-MG mono-cultures despite the presence of C9orf72 pathological features suggesting that a diseased microenvironment may be required to induce phenotypic changes in microglial cells and the associated neuronal dysfunction seen in C9orf72 ALS/FTD neurodegeneration.

Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation.

The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases.

Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS.

Amyotrophic Lateral Sclerosis (ALS), is a fatal neurodegenerative disorder, with TDP-43 inclusions as a major pathological hallmark. Using a Drosophila model of TDP-43 proteinopathy we found significant alterations in glucose metabolism including increased pyruvate, suggesting that modulating glycolysis may be neuroprotective. Indeed, a high sugar diet improves locomotor and lifespan defects caused by TDP-43 proteinopathy in motor neurons or glia, but not muscle, suggesting that metabolic dysregulation occurs in the nervous system. Overexpressing human glucose transporter GLUT-3 in motor neurons mitigates TDP-43 dependent defects in synaptic vesicle recycling and improves locomotion. Furthermore, PFK mRNA, a key indicator of glycolysis, is upregulated in flies and patient derived iPSC motor neurons with TDP-43 pathology. Surprisingly, PFK overexpression rescues TDP-43 induced locomotor deficits. These findings from multiple ALS models show that mechanistically, glycolysis is upregulated in degenerating motor neurons as a compensatory mechanism and suggest that increased glucose availability is protective.