Neurodegenerative diseases (NDs) manifest a wide variety of clinical symptoms depending on the affected brain regions. Gaining insights into why certain regions are resistant while others are susceptible is vital for advancing therapeutic strategies. While gene expression changes offer clues about disease responses across brain regions, the mixture of cell types therein obscures experimental results. In recent years, methods that analyze the transcriptomes of individual cells (e.g., single-cell RNA sequencing or scRNAseq) have been widely used and have provided invaluable insights into specific cell types. Concurrently, transgene-based techniques that dissect cell type-specific translatomes (CSTs) in model systems, like RiboTag and bacTRAP, offer unique advantages but have received less attention. This review juxtaposes the merits and drawbacks of both methodologies, focusing on the use of CSTs in understanding conditions like amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Alzheimer's disease (AD), and specific prion diseases like fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and acquired prion disease. We conclude by discussing the emerging trends observed across multiple diseases and emerging methods.

Although Huntingtons disease (HD) is classically defined by the selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting primarily of granule cells, commonly considered disease resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected in these neurons. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.

Early-life stress can result in life-long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. Here, we show that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher-order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer-1 mutant, we show that the stress-induced decrease in one of these genes, the insulator-binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV w(m4h) reporter. We propose that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.

Thackray et al. report that increased activity of the Hsp70 protein disaggregase system clears human prions and their associated neurotoxicity in variant CJD-infected human PrP transgenic Drosophila. These observations establish the Hsp70 protein disaggregase complex as a viable therapeutic target for human prion disease. The metazoan Hsp70 disaggregase protects neurons from proteotoxicity that arises from the accumulation of misfolded protein aggregates. Hsp70 and its co-chaperones disassemble and extract polypeptides from protein aggregates for refolding or degradation. The effectiveness of the chaperone system decreases with age and leads to accumulation rather than removal of neurotoxic protein aggregates. Therapeutic enhancement of the Hsp70 protein disassembly machinery is proposed to counter late-onset protein misfolding neurodegenerative disease that may arise. In the context of prion disease, it is not known whether stimulation of protein aggregate disassembly paradoxically leads to enhanced formation of seeding competent species of disease-specific proteins and acceleration of neurodegenerative disease. Here we have tested the hypothesis that modulation of Hsp70 disaggregase activity perturbs mammalian prion-induced neurotoxicity and prion seeding activity. To do so we used prion protein (PrP) transgenic Drosophila that authentically replicate mammalian prions. RNASeq identified that Hsp70, DnaJ-1 and Hsp110 gene expression was downregulated in prion-exposed PrP Drosophila. We demonstrated that RNAi knockdown of Hsp110 or DnaJ-1 gene expression in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila enhanced neurotoxicity, whereas overexpression mitigated toxicity. Strikingly, prion seeding activity in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila was ablated or reduced by Hsp110 or DnaJ-1 overexpression, respectively. Similar effects were seen in scrapie prion-exposed ovine PrP Drosophila with modified Hsp110 or DnaJ-1 gene expression. These unique observations show that the metazoan Hsp70 disaggregase facilitates the clearance of mammalian prions and that its enhanced activity is a potential therapeutic strategy for human prion disease.