p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR).

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.

An optimized ATAC-seq protocol for genome-wide mapping of active regulatory elements in primary mouse cortical neurons.

ATAC-seq is a versatile, adaptable, and widely adopted technique for mapping open chromatin regions. However, some biological systems, such as primary neurons, present unique challenges to its application. Conventional ATAC-seq would require the dissociation of the primary neurons after plating but dissociating them leads to rapid cell death and major changes in cell state, affecting ATAC-seq results. We have developed this modified ATAC-seq protocol to address this challenge for primary neurons, providing a high-quality and high-resolution accessible chromatin profile. For complete details on the use and execution of this protocol, please refer to Maor-Nof et al. (2021).

NAP (davunetide) preferential interaction with dynamic 3-repeat Tau explains differential protection in selected tauopathies.

The microtubule (MT) associated protein Tau is instrumental for the regulation of MT assembly and dynamic instability, orchestrating MT-dependent cellular processes. Aberration in Tau post-translational modifications ratio deviation of spliced Tau isoforms 3 or 4 MT binding repeats (3R/4R) have been implicated in neurodegenerative tauopathies. Activity-dependent neuroprotective protein (ADNP) is vital for brain formation and cognitive function. ADNP deficiency in mice causes pathological Tau hyperphosphorylation and aggregation, correlated with impaired cognitive functions. It has been previously shown that the ADNP-derived peptide NAP protects against ADNP deficiency, exhibiting neuroprotection, MT interaction and memory protection. NAP prevents MT degradation by recruitment of Tau and end-binding proteins to MTs and expression of these proteins is required for NAP activity. Clinically, NAP (davunetide, CP201) exhibited efficacy in prodromal Alzheimer's disease patients (Tau3R/4R tauopathy) but not in progressive supranuclear palsy (increased Tau4R tauopathy). Here, we examined the potential preferential interaction of NAP with 3R vs. 4R Tau, toward personalized treatment of tauopathies. Affinity-chromatography showed that NAP preferentially interacted with Tau3R protein from rat brain extracts and fluorescence recovery after photobleaching assay indicated that NAP induced increased recruitment of human Tau3R to MTs under zinc intoxication, in comparison to Tau4R. Furthermore, we showed that NAP interaction with tubulin (MTs) was inhibited by obstruction of Tau-binding sites on MTs, confirming the requirement of Tau-MT interaction for NAP activity. The preferential interaction of NAP with Tau3R may explain clinical efficacy in mixed vs. Tau4R pathologies, and suggest effectiveness in Tau3R neurodevelopmental disorders.

Axonal Degeneration Is Regulated by a Transcriptional Program that Coordinates Expression of Pro- and Anti-degenerative Factors.

Developmental neuronal cell death and axonal elimination are controlled by transcriptional programs, of which their nature and the function of their components remain elusive. Here, we identified the dual specificity phosphatase Dusp16 as part of trophic deprivation-induced transcriptome in sensory neurons. Ablation of Dusp16 enhanced axonal degeneration in response to trophic withdrawal, suggesting that it has a protective function. Moreover, axonal skin innervation was severely reduced while neuronal elimination was increased in the Dusp16 knockout. Mechanistically, Dusp16 negatively regulates the transcription factor p53 and antagonizes the expression of the pro-degenerative factor, Puma (p53 upregulated modulator of apoptosis). Co-ablation of Puma with Dusp16 protected axons from rapid degeneration and specifically reversed axonal innervation loss early in development with no effect on neuronal deficits. Overall, these results reveal that physiological axonal elimination is regulated by a transcriptional program that integrates regressive and progressive elements and identify Dusp16 as a new axonal preserving factor.

Neurite pruning and neuronal cell death: spatial regulation of shared destruction programs.

During development, neurons are initially overproduced and excess neurons are eliminated later on by programmed cell death. In a more refined developmental process termed pruning, excess axons and dendritic branches are removed while the cell body remains intact. In mature animals, axons that become disconnected as a result of injury are eliminated through a series of events collectively known as Wallerian degeneration. Recent evidence points to unexpected similarities between these three types of obliterative processes, as they share common regulators. These findings provide new ideas on how cellular destruction programs are spatially regulated in neurons.

Axonal pruning is actively regulated by the microtubule-destabilizing protein kinesin superfamily protein 2A.

Extensive axonal pruning and neuronal cell death are critical events for the development of the nervous system. Like neuronal cell death, axonal elimination occurs in discrete steps; however, the regulators of these processes remain mostly elusive. Here, we identify the kinesin superfamily protein 2A (KIF2A) as a key executor of microtubule disassembly and axonal breakdown during axonal pruning. Knockdown of Kif2a, but not other microtubule depolymerization or severing proteins, protects axonal microtubules from disassembly upon trophic deprivation. We further confirmed and extended this result to demonstrate that the entire degeneration process is delayed in neurons from the Kif2a knockout mice. Finally, we show that the Kif2a-null mice exhibit normal sensory axon patterning early during development, but abnormal target hyperinnervation later on, as they compete for limited skin-derived trophic support. Overall, these findings reveal a central regulatory mechanism of axonal pruning during development.