Allen Brain Atlas (ABA) provides a valuable resource of spatial/temporal gene expressions in mammalian brains. Despite rich information extracted from this database, current analyses suffer from several limitations. First, most studies are either gene-centric or region-centric, thus are inadequate to capture the superposition of multiple spatial-temporal patterns. Second, standard tools of expression analysis such as matrix factorization can capture those patterns but do not explicitly incorporate spatial dependency. To overcome those limitations, we proposed a computational method to detect recurrent patterns in the spatial-temporal gene expression data of developing mouse brains. We demonstrated that regional distinction in brain development could be revealed by localized gene expression patterns. The patterns expressed in the forebrain, medullary and pontomedullary, and basal ganglia are enriched with genes involved in forebrain development, locomotory behavior, and dopamine metabolism respectively. In addition, the timing of global gene expression patterns reflects the general trends of molecular events in mouse brain development. Furthermore, we validated functional implications of the inferred patterns by showing genes sharing similar spatial-temporal expression patterns with Lhx2 exhibited differential expression in the embryonic forebrains of Lhx2 mutant mice. These analysis outcomes confirm the utility of recurrent expression patterns in studying brain development.

Establishing a balance between excitation and inhibition is critical for brain functions. However, how inhibitory interneurons (INs) generated in the ventral telencephalon integrate with the excitatory neurons generated in the dorsal telencephalon remains elusive. Previous studies showed that INs migrating tangentially to enter the neocortex (NCx), remain in the migratory stream for days before invading the cortical plate during late corticogenesis. Here we show that in developing mouse cortices, INs in the piriform cortex (PCx; the major olfactory cortex) distribute differently from those in the NCx. We provide evidence that during development INs invade and mature earlier in PCx than in NCx, likely owing to the lack of CXCR4 expression in INs from PCx compared to those in NCx. We analyzed IN distribution patterns in Lhx2 cKO mice, where projection neurons in the lateral NCx are re-fated to generate an ectopic PCx (ePCx). The PCx-specific IN distribution patterns found in ePCx suggest that properties of PCx projection neurons regulate IN distribution. Collectively, our results show that the timing of IN invasion in the developing PCx fundamentally differs from what is known in the NCx. Further, our results suggest that projection neurons instruct the PCx-specific pattern of IN distribution.

Axonal transport, an ATP demanding process, plays a critical role in maintaining axonal and neuronal health. ATP in neurons is synthesized by glycolysis and mitochondrial respiration, both driven by proper NAD+/NADH redox potentials. NMNAT2 is the major neuronal NAD+ synthesizing enzyme and is a key axonal maintenance factor. Here, we show that NMNAT2 co-migrates with fast vesicular cargos in axons and is required for fast axonal transport in the distal axons of cortical neurons. Using SoNar sensor imaging to detect axonal NAD+ and NADH, we show that NMNAT2 is critical in maintaining NAD+/NADH potentials in distal axons. With Syn-ATP sensor imaging to detect synaptic vesicle ATP levels (sv-ATP), we demonstrate that glycolysis is the major provider of sv-ATP and NMNAT2 deletion significantly reduces sv-ATP levels. NAD+ supplementation to NMNAT2 KO neurons restores sv-ATP levels and fast axonal transports in a glycolysis-dependent manner. Together, these data show that NMNAT2 maintains the local NAD+/NADH redox potential and sustains on-board glycolysis to meet the bioenergetic demands of fast vesicular transport in distal axons. Intriguingly, mitochondrial respiration contributes significantly to sv-ATP levels in NMNAT2 KO axons. This finding suggests that NMNAT2 deletion induces metabolic plasticity by engaging mitochondria. Surprisingly, supplying NMN, the substrate for NMNAT2 in NAD+ synthesis, restores sv-ATP and axonal transport in NMNAT2 KO axons with an efficacy similar to NAD+. The restoration of NMNAT2 KO distal axonal sv-ATP levels and vesicle transport by NMN requires mitochondrial respiration, while the rescue by NAD+ is independent of mitochondrial respiration. Based on these findings, we hypothesize that NMN rescues glycolysis to restore axonal transport in NMNAT2 KO axons by taking advantage of the compensatory ATP-generating metabolic pathways triggered by NMNAT2 loss.

Background Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson's, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function. Methods We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos. Results We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons. Conclusion NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Here we show that deleting NMNAT2 from cortical glutamatergic neurons (NMNAT2 cKO) results in progressive axonal loss, neuroinflammation, small hippocampi and enlarged ventricles. Interestingly, dramatic neuroinflammation responses were observed around the long-range axonal tracts of NMNAT2 cKO cortical neurons. In addition to the neurodegenerative-like phenotype, we also found the absence of whisker-representation patterns (barrels) in the primary somatosensory cortex of NMNAT2 cKO mice. These observations suggest that NMNAT2 is required in developing cortical circuits and in maintaining the health of cortical neurons. Unbiased transcriptomic analysis suggests that NMNAT2 loss in cortical neurons after axonal outgrowth phase upregulates mitochondria function while greatly reducing synaptogenesis pathways. Complete loss of Sarm1 function in NMNAT2 cKO mice restores barrel map formation and axonal integrity and abolishes the inflammatory response. Interestingly, reducing Sarm1 function in NMNAT2 cKO mice by deleting only one copy of Sarm1 restores barrel map formation but did not diminish the neurodegenerative-like phenotype. Only complete loss of Sarm1 prevents neurodegeneration and inflammatory responses.