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Repetitive physical insults to the head, including those that elicit mild traumatic brain injury (mTBI), are a known risk factor for a variety of neurodegenerative conditions including Alzheimer’s disease (AD), Parkinson’s disease (PD), and chronic traumatic encephalopathy (CTE). Although most individuals who sustain mTBI typically achieve a seemingly full recovery within a few weeks, a subset experience delayed-onset symptoms later in life. As most mTBI research has focused on the acute phase of injury, there is an incomplete understanding of mechanisms related to the late-life emergence of neurodegeneration after early exposure to mild head trauma. The recent adoption of Drosophila-based brain injury models provides several unique advantages over existing preclinical animal models, including a tractable framework amenable to high-throughput assays and short relative lifespan conducive to lifelong mechanistic investigation. The use of flies also provides an opportunity to investigate important risk factors associated with neurodegenerative conditions, specifically age and sex. In this review, we survey current literature that examines age and sex as contributing factors to head trauma-mediated neurodegeneration in humans and preclinical models, including mammalian and Drosophila models. We discuss similarities and disparities between human and fly in aging, sex differences, and pathophysiology. Finally, we highlight Drosophila as an effective tool for investigating mechanisms underlying head trauma-induced neurodegeneration and for identifying therapeutic targets for treatment and recovery.

Dendritic spines are small actin-rich protrusions essential for the formation of functional circuits in the mammalian brain. During development, spines begin as dynamic filopodia-like protrusions that are then replaced by relatively stable spines containing an expanded head. Remodeling of the actin cytoskeleton plays a key role in the formation and modification of spine morphology, however many of the underlying regulatory mechanisms remain unclear. Capping protein (CP) is a major actin regulating protein that caps the barbed ends of actin filaments, and promotes the formation of dense branched actin networks. Knockdown of CP impairs the formation of mature spines, leading to an increase in the number of filopodia-like protrusions and defects in synaptic transmission. Here, we show that CP promotes the stabilization of dendritic protrusions, leading to the formation of stable mature spines. However, the localization and function of CP in dendritic spines requires interactions with proteins containing a capping protein interaction (CPI) motif. We found that the CPI motif-containing protein Twinfilin-1 (Twf1) also localizes to spines where it plays a role in CP spine enrichment. The knockdown of Twf1 leads to an increase in the density of filopodia-like protrusions and a decrease in the stability of dendritic protrusions, similar to CP knockdown. Finally, we show that CP directly interacts with Shank and regulates its spine accumulation. These results suggest that spatiotemporal regulation of CP in spines not only controls the actin dynamics underlying the formation of stable postsynaptic spine structures, but also plays an important role in the assembly of the postsynaptic apparatus underlying synaptic function.

Abstract Mild head trauma, including concussion, can lead to chronic brain dysfunction and degeneration but the underlying mechanisms remain poorly understood. Here, we developed a novel head impact system to investigate the long-term effects of mild head trauma on brain structure and function, as well as the underlying mechanisms in Drosophila melanogaster. We find that Drosophila subjected to repetitive head impacts develop long-term deficits, including impaired startle-induced climbing, progressive brain degeneration, and shortened lifespan, all of which are substantially exacerbated in female flies. Interestingly, head impacts elicit an elevation in neuronal activity and its acute suppression abrogates the detrimental effects in female flies. Together, our findings validate Drosophila as a suitable model system for investigating the long-term effects of mild head trauma, suggest an increased vulnerability to brain injury in female flies, and indicate that early altered neuronal excitability may be a key mechanism linking mild brain trauma to chronic degeneration.

Summary: Drosophila melanogaster is an excellent model organism to study neurodegeneration. Assessing evident neurodegeneration within the fly brain involves the laborious preparation of thin-sectioned H&E-stained heads to visualize brain vacuole degeneration. Here, we present an advanced microscopy-based protocol, without the need for sectioning, to detect vacuole degeneration within whole fly brains by applying commonly used stains to reveal the brain parenchyma. This approach preserves the whole-brain architecture and enables rapid, reproducible, and quantitative analyses of vacuole-like degeneration associated with specific brain regions.For complete details on the use and execution of this protocol, please refer to Behnke et al. (2021).

The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.

Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer-binding protein thymosin β4 (Tβ4) for optimal leading-edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it does not interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions.

Environmental insults, including mild head trauma, significantly increase the risk of neurodegeneration. However, it remains challenging to establish a causative connection between early-life exposure to mild head trauma and late-life emergence of neurodegenerative deficits, nor do we know how sex and age compound the outcome. Using a Drosophila model, we demonstrate that exposure to mild head trauma causes neurodegenerative conditions that emerge late in life and disproportionately affect females. Increasing age-at-injury further exacerbates this effect in a sexually dimorphic manner. We further identify sex peptide signaling as a key factor in female susceptibility to post-injury brain deficits. RNA sequencing highlights a reduction in innate immune defense transcripts specifically in mated females during late life. Our findings establish a causal relationship between early head trauma and late-life neurodegeneration, emphasizing sex differences in injury response and the impact of age-at-injury. Finally, our findings reveal that reproductive signaling adversely impacts female response to mild head insults and elevates vulnerability to late-life neurodegeneration.

We exposed a headform instrumented with 10 pressure sensors mounted flush with the surface to a shock wave with three nominal intensities: 70, 140 and 210 kPa. The headform was mounted on a Hybrid III neck, in a rigid configuration to eliminate motion and associated pressure variations. We evaluated the effect of the test location by placing the headform inside, at the end and outside of the shock tube. The shock wave intensity gradually decreases the further it travels in the shock tube and the end effect degrades shock wave characteristics, which makes comparison of the results obtained at three locations a difficult task. To resolve these issues, we developed a simple strategy of data reduction: the respective pressure parameters recorded by headform sensors were divided by their equivalents associated with the incident shock wave. As a result, we obtained a comprehensive set of non-dimensional parameters. These non-dimensional parameters (or amplification factors) allow for direct comparison of pressure waveform characteristic parameters generated by a range of incident shock waves differing in intensity and for the headform located in different locations. Using this approach, we found a correlation function which allows prediction of the peak pressure on the headform that depends only on the peak pressure of the incident shock wave (for specific sensor location on the headform), and itis independent on the headform location. We also found a similar relationship for the rise time. However, for the duration and impulse, comparable correlation functions do not exist. These findings using a headform with simplified geometry are baseline values and address a need for the development of standardized parameters for the evaluation of personal protective equipment (PPE) under shock wave loading.