Histone deacetylases (HDACs) reduce acetylation of histones and other proteins. HDAC inhibition has been explored as a treatment to promote neural repair in the brain. In rodents, HDAC6 inhibition boosted neurite outgrowth and survival, but its role in human neural repair remains unclear. To directly assess the role of HDAC6 in neurite outgrowth, we used CRISPR/Cas-9 to knock out HDAC6 (KO) in a human embryonic stem cell line harboring inducible NGN2, a transcription factor that rapidly induces progenitor cells to differentiate into neurons. NGN2-induced cortical neurons (iNeurons) were derived from these ESCs. Preliminary results yielded mixed results on neurite outgrowth, with some data showing increased effects with HDAC6 KO iNeurons on inhibitory conditions and other data revealing no significant difference on both permissive and inhibitory conditions; further experimentation is required. Control induced neurons will also be treated with the broad spectrum HDAC inhibitors (belinostat, panobinostat, and valproic acid), as well as a relatively selective HDAC6 inhibitor (tubastatin A), to determine varying inhibition effects on neurite outgrowth. Previous data showed that broad spectrum HDAC inhibitors do not significantly increase acetylated α-tubulin levels, a target of HDAC6, compared to tubastatin A, suggesting that effects of the pan-HDAC inhibition drugs may be independent of HDAC6. Future steps would involve determining a consistent trend of HDAC6 KO, HDAC(6) inhibition, and their combination on neurite outgrowth.

Human genetic studies identified 70+ genetic loci associated with modified risk for Alzheimer's disease (AD). Over a third of the genes linked to AD modifier loci are selectively expressed in microglia. Among microglia-enriched AD GWAS genes, the R47H variant of TREM2 has been identified as one of the strongest AD-risk alleles. In contrast, the P522R variant of PLCG2 confers a significant protective effect against AD. Given the critical role of TREM2 signaling, we studied key component proteins in this pathway by crossing 5xFAD with Plcg2-522R knock-in (KI), Dap10 knock-out (KO), Dap12-KO, and Dap10/Dap12 double KO mouse lines. We found that expressing Plcg2-522R reduced amyloid burden in 5xFAD. Consistent with the RNA-sequencing result, C1q in situ hybridization (RNAscope) showed a similar trend of increased expression in Plcg2-P522R/5xFAD mice. In contrast, KO either or both TREM2 binding partners adversely increased amyloid deposition and worsened neuropathology in AD mouse models. We utilized primary microglia to investigate the molecular mechanism of how PLCG2 and TREM2 modify microglia responses to fAβ. The upregulation of TREM2 (both WT and R47H alleles) and expressing Plcg2-522R appeared to alter microglial expression of the genes related to microglial survival (Csf1r), inflammatory (Il-1β) and phagocytosis (Spp1) in response to fAβ. This study provides a molecular insight into the role of TREM2 signaling in AD pathogenesis and in regulating microglial function, which may help to identify therapeutic targets for AD.

Increased sleep is vital for neural injury recovery. While sleep facilitates neural debris clearance, the underlying mechanisms remain unclear. Since highwire regulates Wallerian degeneration, we examined how downstream targets influence sleep and axon clearance post-injury. We found that overexpression of the neuroprotective transgenes, nmnat2 and wallenda, elevates sleep pressure after antennal injury. While wild-type flies show an increase in sleep acutely following injury for 24 hours, flies overexpressing nmnat2 or wallenda maintain high levels of sleep for at least 72 hours. Day sleep bout length and day bout number were higher in wallenda relative to controls for 48-72 hours post-injury. Next, confocal imaging was performed to quantify synapse removal at different time points post-injury. Overexpressing nmnat2 in olfactory receptor neurons retained more presynaptic active zone protein Bruchpilot (BRP) than wild-type flies at 6 and 24 hours post-injury while overexpressing wallenda resulted in near complete loss of BRP for the uninjured controls. Our results show that each gene distinctly impacts synaptic abundance and removal post-injury. Lastly, we show that innate immune response marker Spätzle is expressed twofold in nmnat2 6 hours post-injury relative to wild-type flies. Further research into glial-neuronal crosstalk may reveal links between immune function and chronic increased sleep. This has implications for neurodegenerative diseases by understanding sleep’s impact on axonal health under cellular stress.

Oligodendrocytes produce extensive myelin membranes to insulate axons. As axons and myelin expand during development, the oligodendrocytes' plasma membranes undergo physical stretching. In multiple sclerosis, the complement membrane attack complex forms pores in the myelin membrane. Given the challenges to plasma membrane integrity, maintaining this integrity is vital for oligodendrocyte functionality and survival, although the underlying mechanisms are unknown. We hypothesized that VAMP2/3-dependent exocytosis actively repairs plasma membrane damage in mature oligodendrocytes. To test this, we employed genetic tools to inhibit VAMP2/3-dependent exocytosis in these cells. Our findings indicate that inhibiting VAMP2/3-dependent exocytosis compromises oligodendrocyte membrane repair following laser-induced membrane injury in vitro. Intriguingly, blocking oligodendrocyte exocytosis in vivo results in the prolonged reactivity of microglia and astrocytes. Ongoing studies aim to determine whether oligodendrocyte membrane integrity is compromised in vivo and how oligodendrocyte exocytosis contributes to maintaining the homeostatic state of microglia and astrocytes. This research sheds light on the underexplored domain of myelin plasma membrane repair, revealing potential therapeutic targets for demyelinating disorders. Additionally, it unveils new insights into the interactions between oligodendrocytes, microglia, and astrocytes.

Charcot-Marie-Tooth Disease (CMT) is a genetic neuropathy marked by progressive muscle weakness, slowed nerve conduction, and myelin abnormalities. The CMT4B1 subtype is caused by mutations in the MTMR2 gene, which impairs its phosphatase activity on PI(3,5)P2, a lipid crucial for regulating endosomal trafficking. This process is essential for transporting and recycling synaptic proteins, especially in excitatory neurons that rely on stable protein turnover for signaling. This project investigates MTMR2’s role in proprioceptive DRG neurons that synapse onto the spinal cord, focusing on endosomal trafficking. MTMR2 is known to support excitatory synapse maintenance in the hippocampus through its interaction with PSD-95, a postsynaptic scaffolding protein that anchors and organizes synaptic signaling complexes. Despite observed deficits in proprioceptive neurons of Mtmr2 mutants, its role in spinal circuits remains unclear. We will label spinal cord sections from MTMR2 flox mice with synaptophysin (presynaptic) and PSD-95 (postsynaptic) markers. Colocalization and synapse morphology will be quantified using ImageJ and Synbot, with thresholding done manually or via Ilastik, a machine learning-based model. We expect to observe reduced colocalization of synaptophysin and PSD-95, altered synapse morphology, and decreased marker intensity in MTMR2 flox mutants indicating impaired excitatory synapse maintenance due to disrupted endosomal trafficking in proprioceptive neurons.