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Summary: Studies of human induced pluripotent stem cell (iPSC)-derived neurons promise important insights into neurodegenerative diseases. Here, we present a protocol for live imaging of axonal transport in glutamatergic iPSC-derived neurons (iNeurons). We describe steps for the differentiation of iPSCs into iNeurons via PiggyBac-mediated neurogenin 2 (NGN2) delivery, iNeuron culture and transfection, and the acquisition and analysis of time-lapse images. Our protocol is optimized for the widely available catalog of KOLF2.1J iPSCs with mutations relevant to neurodegenerative diseases but is also applicable to other iPSC lines.For complete details on the use and execution of this protocol, please refer to Dou et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Gain-of-function mutations in the LRRK2 gene cause Parkinson’s disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We find that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin. In iPSC-derived human neurons, knockin of the strongly hyperactive LRRK2-p.R1441H mutation causes striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knockout of the opposing protein phosphatase 1H (PPM1H) phenocopies the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuates transport defects in both p.R1441H knockin and PPM1H knockout neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive “tug-of-war” between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy.

Summary: In response to lysosomal damage, cells engage several quality-control mechanisms, including the selective isolation and degradation of damaged lysosomes by lysophagy. Here, we report that the selective autophagy adaptor SQSTM1/p62 is recruited to damaged lysosomes in both HeLa cells and neurons and is required for lysophagic flux. The Phox and Bem1p (PB1) domain of p62 mediates oligomerization and is specifically required for lysophagy. Consistent with this observation, we find that p62 forms condensates on damaged lysosomes. These condensates are precisely tuned by the small heat shock protein HSP27, which is phosphorylated in response to lysosomal injury and maintains the liquidity of p62 condensates, facilitating autophagosome formation. Mutations in p62 have been identified in patients with amyotrophic lateral sclerosis (ALS); ALS-associated mutations in p62 impair lysophagy, suggesting that deficits in this pathway may contribute to neurodegeneration. Thus, p62 condensates regulated by HSP27 promote lysophagy by forming platforms for autophagosome biogenesis at damaged lysosomes.

Summary: Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis.

Amyotrophic lateral sclerosis (ALS) is a debilitating and incurable disease involving the loss of motor neurons and subsequent muscle atrophy. Genetic studies have implicated deficits in autophagy and/or mitophagy in the onset of the disease. Here we review recent progress in our understanding of the pathways for autophagy and mitophagy in neurons, and how these pathways may be affected by mutations in genes including DCTN1, OPTN, TBK1, VCP, and C9ORF72. We also discuss the implications of modulating autophagy in ALS, highlighting both the potential of the approach and the concerns raised by targeting this pathway as a therapeutic strategy in neurodegenerative disease.

The kinesin KIF21B is implicated in several human neurological disorders, including delayed cognitive development, yet it remains unclear how KIF21B dysfunction may contribute to pathology. One limitation is that relatively little is known about KIF21B-mediated physiological functions. Here, we generated Kif21b knockout mice and used cellular assays to investigate the relevance of KIF21B in neuronal and in vivo function. We show that KIF21B is a processive motor protein and identify an additional role for KIF21B in regulating microtubule dynamics. In neurons lacking KIF21B, microtubules grow more slowly and persistently, leading to tighter packing in dendrites. KIF21B-deficient neurons exhibit decreased dendritic arbor complexity and reduced spine density, which correlate with deficits in synaptic transmission. Consistent with these observations, Kif21b-null mice exhibit behavioral changes involving learning and memory deficits. Our study provides insight into the cellular function of KIF21B and the basis for cognitive decline resulting from KIF21B dysregulation.

Motor-cargo recruitment to microtubules is often the rate-limiting step of intracellular transport, and defects in this recruitment can cause neurodegenerative disease. Here, we use in vitro reconstitution assays with single-molecule resolution, live-cell transport assays in primary neurons, computational image analysis, and computer simulations to investigate the factors regulating retrograde transport initiation in the distal axon. We find that phosphorylation of the cytoskeletal-organelle linker protein CLIP-170 and post-translational modifications of the microtubule track combine to precisely control the initiation of retrograde transport. Computer simulations of organelle dynamics in the distal axon indicate that while CLIP-170 primarily regulates the time to microtubule encounter, the tyrosination state of the microtubule lattice regulates the likelihood of binding. These mechanisms interact to control transport initiation in the axon in a manner sensitive to the specialized cytoskeletal architecture of the neuron.

Axonal transport is essential for neuronal function, and defects in transport are associated with multiple neurodegenerative diseases. Aberrant cyclin-dependent kinase 5 (CDK5) activity, driven by the stress-induced activator p25, also is observed in these diseases. Here we show that elevated CDK5 activity increases the frequency of nonprocessive events for a range of organelles, including lysosomes, autophagosomes, mitochondria, and signaling endosomes. Transport disruption induced by aberrant CDK5 activation depends on the Lis1/Ndel1 complex, which directly regulates dynein activity. CDK5 phosphorylation of Ndel1 favors a high affinity Lis1/Ndel/dynein complex that blocks the ATP-dependent release of dynein from microtubules, inhibiting processive motility of dynein-driven cargo. Similar transport defects observed in neurons from a mouse model of amyotrophic lateral sclerosis are rescued by CDK5 inhibition. Together, these studies identify CDK5 as a Lis1/Ndel1-dependent regulator of transport in stressed neurons, and suggest that dysregulated CDK5 activity contributes to the transport deficits observed during neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease leading to motor neuron cell death, but recent studies suggest that non-neuronal cells may contribute to the pathological mechanisms involved. Myostatin is a negative regulator of muscle growth whose function can be inhibited using neutralizing antibodies. In this study, we used transgenic mouse and rat models of ALS to test whether treatment with anti-myostatin antibody slows muscle atrophy, motor neuron loss, or disease onset and progression. Significant increases in muscle mass and strength were observed in myostatin-antibody-treated SOD1G93A mice and rats prior to disease onset and during early-stage disease. By late stage disease, only diaphragm muscle remained significantly different in treated animals in comparison to untreated controls. Myostatin inhibition did not delay disease onset nor extend survival in either the SOD1G93A mouse or rat. Together, these results indicate that inhibition of myostatin does not protect against the onset and progression of motor neuron degenerative disease. However, the preservation of skeletal muscle during early-stage disease and improved diaphragm morphology and function maintained through late stage disease suggest that anti-myostatin therapy may promote some improved muscle function in ALS.