Postsynaptic BMP signaling regulates myonuclear properties in Drosophila larval muscles.

The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that then positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity.

Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Our previous work has shown Drosophila cofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA sequencing analysis which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand our understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology.

The effect of amyloid on microglia-neuron interactions before plaque onset occurs independently of TREM2 in a mouse model of Alzheimer's disease.

Genetic studies identified mutations in several immune-related genes that confer increased risk for developing Alzheimer's disease (AD), suggesting a key role for microglia in AD pathology. Microglia are recruited to and actively modulate the local toxicity of amyloid plaques in models of AD through these cells' transcriptional and functional reprogramming to a disease-associated phenotype. However, it remains unknown whether microglia actively respond to amyloid accumulation before plaque deposition in AD. We compared microglial interactions with neurons that exhibit amyloid accumulation to those that do not in 1-month-old 5XFAD mice to determine which aspects of microglial morphology and function are altered by early 6E10+ amyloid accumulation. We provide evidence of preferential microglial process engagement of amyloid laden neurons. Microglia, on exposure to amyloid, also increase their internalization of neurites even before plaque onset. Unexpectedly, we found that triggering receptor expressed on myeloid cells 2 (TREM2), which is critical for microglial responses to amyloid plaque pathology later in disease, is not required for enhanced microglial interactions with neurons or neurite internalization early in disease. However, TREM2 was still required for early morphological changes exhibited by microglia. These data demonstrate that microglia sense and respond to amyloid accumulation before plaques form using a distinct mechanism from the TREM2-dependent pathway required later in disease.

TREM2 is required for microglial instruction of astrocytic synaptic engulfment in neurodevelopment.

Variants in the microglial receptor TREM2 confer risk for multiple neurodegenerative diseases. However, it remains unknown how this receptor functions on microglia to modulate these diverse neuropathologies. To understand the role of TREM2 on microglia more generally, we investigated changes in microglial function in Trem2-/- mice. We found that loss of TREM2 impairs normal neurodevelopment, resulting in reduced synapse number across the cortex and hippocampus in 1-month-old mice. This reduction in synapse number was not due directly to alterations in interactions between microglia and synapses. Rather, TREM2 was required for microglia to limit synaptic engulfment by astrocytes during development. While these changes were largely normalized later in adulthood, high fat diet administration was sufficient to reinitiate TREM2-dependent modulation of synapse loss. Together, this identifies a novel role for microglia in instructing synaptic pruning by astrocytes to broadly regulate appropriate synaptic refinement, and suggests novel candidate mechanisms for how TREM2 and microglia could influence synaptic loss in brain injury and disease.

The Trem2 R47H variant confers loss-of-function-like phenotypes in Alzheimer's disease.

BACKGROUND: The R47H variant of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) confers greatly increased risk for Alzheimer's disease (AD), reflective of a central role for myeloid cells in neurodegeneration. Understanding how this variant confers AD risk promises to provide important insights into how myeloid cells contribute to AD pathogenesis and progression. METHODS: In order to investigate this mechanism, CRISPR/Cas9 was used to generate a mouse model of AD harboring one copy of the single nucleotide polymorphism (SNP) encoding the R47H variant in murine Trem2. TREM2 expression, myeloid cell responses to amyloid deposition, plaque burden, and neuritic dystrophy were assessed at 4 months of age. RESULTS: AD mice heterozygous for the Trem2 R47H allele exhibited reduced total Trem2 mRNA expression, reduced TREM2 expression around plaques, and reduced association of myeloid cells with plaques. These results were comparable to AD mice lacking one copy of Trem2. AD mice heterozygous for the Trem2 R47H allele also showed reduced myeloid cell responses to amyloid deposition, including a reduction in proliferation and a reduction in CD45 expression around plaques. Expression of the Trem2 R47H variant also reduced dense core plaque number but increased plaque-associated neuritic dystrophy. CONCLUSIONS: These data suggest that the AD-associated TREM2 R47H variant increases risk for AD by conferring a loss of TREM2 function and enhancing neuritic dystrophy around plaques.

Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells.

α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS '3K' mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and '3K' further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.

TREM2 in Neurodegenerative Diseases.

TREM2 variants have been identified as risk factors for Alzheimer's disease (AD) and other neurodegenerative diseases (NDDs). Because TREM2 encodes a receptor exclusively expressed on immune cells, identification of these variants conclusively demonstrates that the immune response can play an active role in the pathogenesis of NDDs. These TREM2 variants also confer the highest risk for developing Alzheimer's disease of any risk factor identified in nearly two decades, suggesting that understanding more about TREM2 function could provide key insights into NDD pathology and provide avenues for novel immune-related NDD biomarkers and therapeutics. The expression, signaling and function of TREM2 in NDDs have been extensively investigated in an effort to understand the role of immune function in disease pathogenesis and progression. We provide a comprehensive review of our current understanding of TREM2 biology, including new insights into the regulation of TREM2 expression, and TREM2 signaling and function across NDDs. While many open questions remain, the current body of literature provides clarity on several issues. While it is still often cited that TREM2 expression is decreased by pro-inflammatory stimuli, it is now clear that this is true in vitro, but inflammatory stimuli in vivo almost universally increase TREM2 expression. Likewise, while TREM2 function is classically described as promoting an anti-inflammatory phenotype, more than half of published studies demonstrate a pro-inflammatory role for TREM2, suggesting that its role in inflammation is much more complex. Finally, these components of TREM2 biology are applied to a discussion of how TREM2 impacts NDD pathologies and the latest assessment of how these findings might be applied to immune-directed clinical biomarkers and therapeutics.

KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity.

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into ß-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs ß- and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescent-protein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in buffer-insoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.

Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation.

ß-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.