A neuropathy-associated kinesin KIF1A mutation hyper-stabilizes the motor-neck interaction during the ATPase cycle.

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the ß7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on ß7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.

F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain.

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.

Insight into the regulation of axonal transport from the study of KIF1A-associated neurological disorder.

Neuronal function depends on axonal transport by kinesin superfamily proteins (KIFs). KIF1A is the molecular motor that transports synaptic vesicle precursors, synaptic vesicles, dense core vesicles and active zone precursors. KIF1A is regulated by an autoinhibitory mechanism; many studies, as well as the crystal structure of KIF1A paralogs, support a model whereby autoinhibited KIF1A is monomeric in solution, whereas activated KIF1A is dimeric on microtubules. KIF1A-associated neurological disorder (KAND) is a broad-spectrum neuropathy that is caused by mutations in KIF1A. More than 100 point mutations have been identified in KAND. In vitro assays show that most mutations are loss-of-function mutations that disrupt the motor activity of KIF1A, whereas some mutations disrupt its autoinhibition and abnormally hyperactivate KIF1A. Studies on disease model worms suggests that both loss-of-function and gain-of-function mutations cause KAND by affecting the axonal transport and localization of synaptic vesicles. In this Review, we discuss how the analysis of these mutations by molecular genetics, single-molecule assays and force measurements have helped to reveal the physiological significance of KIF1A function and regulation, and what physical parameters of KIF1A are fundamental to axonal transport.

De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors.

KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along microtubules. Mutations in human Kif1a lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established Caenorhabditis elegans models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our C. elegans models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.

Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head-bound state.

KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. However, structural features underlying this function are unclear. Here, we determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine-rich insertion in loop-12 termed the 'K-loop', which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native KIF1A loop-12 with that of kinesin-1 resulted in a 6-fold decrease in run length, whereas adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell time assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant than WT. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing binding of the motor in its vulnerable one-head-bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that KIF1A processivity is linearly dependent on the charge of loop-12, further highlighting how loop-12 contributes to the function of this key motor protein.

Heterogeneity of comprehensive clinical phenotype and longitudinal adaptive function and correlation with computational predictions of severity of missense genotypes in KIF1A-associated neurological disorder.

PURPOSE: Pathogenic variants in kinesin family member 1A (KIF1A) are associated with KIF1A-associated neurological disorder. We report the clinical phenotypes and correlate genotypes of individuals with KIF1A-associated neurological disorder. METHODS: Medical history and adaptive function were assessed longitudinally. In-person evaluations included neurological, motor, ophthalmologic, and cognitive assessments. RESULTS: We collected online data on 177 individuals. Fifty-seven individuals were also assessed in-person. Most individuals had de novo heterozygous missense likely pathogenic/pathogenic KIF1A variants. The most common characteristics were hypotonia, spasticity, ataxia, seizures, optic nerve atrophy, cerebellar atrophy, and cognitive impairment. Mean Vineland adaptive behavior composite score (VABS-ABC) was low (M = 62.9, SD = 19.1). The mean change in VABS-ABC over time was -3.1 (SD = 7.3). The decline in VABS-ABC was associated with the age at first assessment and abnormal electroencephalogram/seizure. There was a positive correlation between evolutionary scale model (ESM) score for the variants and final VABS-ABC (P = .003). Abnormal electroencephalogram/seizure, neuroimaging result, and ESM explain 34% of the variance in final VABS-ABC (P < .001). CONCLUSION: In-person assessment confirmed caregiver report and identified additional visual deficits. Adaptive function declined over time consistent with both the neurodevelopmental and neurodegenerative nature of the condition. Using ESM score assists in predicting phenotype across a wide range of unique variants.

Long-term clinical observation of patients with heterozygous KIF1A variants.

KIF1A-related disorders (KRDs) encompass recessive and dominant variants with wide clinical variability. Recent genetic investigations have expanded the clinical phenotypes of heterozygous KIF1A variants. However, there have been a few long-term observational studies of patients with heterozygous KIF1A variants. A retrospective chart review of consecutive patients diagnosed with spastic paraplegia at Miyagi Children's Hospital from 2016 to 2020 identified six patients with heterozygous KIF1A variants. To understand the long-term changes in clinical symptoms, we examined these patients in terms of their characteristics, clinical symptoms, results of electrophysiological and neuroimaging studies, and genetic testing. The median follow-up period was 30 years (4-44 years). This long-term observational study showed that early developmental delay and equinus gait, or unsteady gait, are the first signs of disease onset, appearing with the commencement of independent walking. In addition, later age-related progression was observed in spastic paraplegia, and the appearance of axonal neuropathy and reduced visual acuity were characteristic features of the late disease phenotype. Brain imaging showed age-related progression of cerebellar atrophy and the appearance of hyperintensity of optic radiation on T2WI and FLAIR imaging. Long-term follow-up revealed a pattern of steady progression and a variety of clinical symptoms, including spastic paraplegia, peripheral neuropathy, reduced visual acuity, and some degree of cerebellar ataxia. Clinical variability between patients was observed to some extent, and therefore, further studies are required to determine the phenotype-genotype correlation.

A Novel De Novo Missense Mutation in KIF1A Associated with Young-Onset Upper-Limb Amyotrophic Lateral Sclerosis.

We investigate the etiology of amyotrophic lateral sclerosis (ALS) in a 35-year-old woman presenting with progressive weakness in her left upper limb. Prior to sequencing, a comprehensive neurological work-up was performed, including neurological examination, electrophysiology, biomarker assessment, and brain and spinal cord MRI. Six months before evaluation, the patient experienced weakness and atrophy in her left hand, accompanied by brisk reflexes and Hoffman sign in the same arm. Electroneuromyography revealed lower motor neuron involvement in three body regions. Neurofilament light chains were elevated in her cerebrospinal fluid. Brain imaging showed asymmetrical T2 hyperintensity of the corticospinal tracts and T2 linear hypointensity of the precentral gyri. Trio genome sequencing identified a likely pathogenic de novo variant in the KIF1A gene (NM_001244008.2): c.574A>G, p.(Ile192Val). Pathogenic variants in KIF1A have been associated with a wide range of neurological manifestations called KIF1A-associated neurological diseases (KAND). This report describes a likely pathogenic de novo variant in KIF1A associated with ALS, expanding the phenotypic spectrum of KAND and our understanding of the pathophysiology of ALS.

A de novo low-frequency mosaic variant of KIF1A causes hereditary spastic paraplegia: A literature review.

OBJECTIVE: The objective of this study was to investigate the pathogenesis and inheritance pattern of a Chinese Han family with hereditary spastic paraplegia and to retrospectively analyze the characteristics of KIF1A gene variants and related clinical manifestations. METHODS: High-throughput whole-exome sequencing was performed on members of a Chinese Han family with a clinical diagnosis of hereditary spastic paraplegia, and the sequencing results were validated by Sanger sequencing. Deep high-throughput sequencing was performed on subjects with suspected mosaic variants. The previously reported pathogenic variant loci of the KIF1A gene with complete data were collected, and the clinical manifestations and characteristics of the pathogenic KIF1A gene variant were analyzed. RESULTS: A pathogenic heterozygous variant located in the neck coil of the KIF1A gene (c.1139G>C, p.Arg380Pro) was identified in the proband and four additional members of the family. It was derived from the de novo low-frequency somatic-gonadal mosaicism of the proband's grandmother and had a rate of 10.95%. INTERPRETATION: This study helps us to better understand the pathogenic mode and characteristics of mosaic variants, and to understand the location and clinical characteristics of pathogenic variants in KIF1A.

A Novel de novo KIF1A Mutation in a Patient with Ataxia, Intellectual Disability and Mild Foot Deformity.

Early-onset ataxias are often difficult to diagnose due to the genetic and phenotypic heterogeneity of patients. Whole exome sequencing (WES) is a powerful method for determining causative mutations of early-onset ataxias. We report a case in which a novel de novo KIF1A mutation was identified in a patient with ataxia, intellectual disability and mild foot deformity.A patient presented with sporadic forms of ataxia with mild foot deformity, intellectual disability, peripheral neuropathy, pyramidal signs, and orthostatic hypotension. WES was used to identify a novel de novo mutation in KIF1A, a known causative gene of neurodegeneration and spasticity with or without cerebellar atrophy or cortical visual impairment syndrome (NESCAVS).We report a novel phenotype of NESCAVS that is associated with a novel de novo missense mutation in KIF1A, which provides valuable information for the diagnosis of NESCAVS even in the era of WES. Early rehabilitation of patients with NESCAVS may prevent symptom worsening and improve the disease course.

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OBJECTIVES: To analyze the clinical and genetic characteristics of children with autosomal dominant neurodevelopmental disorders caused by kinesin family member 1A (KIF1A) gene variation. METHODS: Clinical and genetic testing data of 6 children with KIF1A gene de novo heterozygous variation diagnosed in Shanghai Children's Medical Center Affiliated to Shanghai Jiao Tong University School of Medicine from the year 2018 to 2020 were retrospectively analyzed. Pathogenic variants were identified based on whole exome sequencing, and verified by Sanger sequencing. Moreover, the effect of variants on three-dimensional structure and stability of protein was analyzed by bioinformatics. RESULTS: Among 6 patients there were 4 males and 2 females, and the age of consultation varied from 7 months to 18 years. All cases had varying degrees of motor developmental delay since childhood, and 4 of them had gait abnormalities or fell easily. In addition, 2 children were accompanied by delayed mental development, epilepsy and abnormal eye development. Genetic tests showed that all 6 cases had heterozygous de novo variations of KIF1A gene, including 4 missense mutations c.296C>T (p.T99M), c.761G>A (p.R254Q), c.326G>T (p.G109V), c.745C>G (p.L249V) and one splicing mutation c.798+1G>A, among which the last three variants have not been previously reported. Bioinformatics analysis showed that G109V and L249V may impair their interaction with the neighboring amino acid residues, thereby impacting protein function and reducing protein stability, and were assessed as "likely pathogenic". Meanwhile, c.798+1G>A may damage an alpha helix in the motor domain of the KIF1A protein, and was assessed as "likely pathogenic". CONCLUSIONS: KIF1A-associated neurological diseases are clinically heterogeneous, with motor developmental delay and abnormal gait often being the most common clinical features. The clinical symptoms in T99M carriers are more severe, while those in R254Q carriers are relatively mild.

Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors.

KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations in KIF1A are associated with the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into the Caenorhabditis elegans KIF1A homolog unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.

KIF1A novel frameshift variant p.(Ser887Profs*64) exhibits clinical heterogeneity in a Pakistani family with hereditary sensory and autonomic neuropathy type IIC.

Background: Hereditary sensory and autonomic neuropathies (HSANs) are rare heterogeneous group of neurological disorders caused by peripheral nerve deterioration. The HSANs sub-clinical classes have clinical and genetic overlap which often lead to misdiagnosis. In the present study a Pakistani family with five affected members suffering from severe neuropathy were genetically analyzed to identify the disease causative element in the family.Methods: Genome wide high-density single nucleotide polymorphism (SNP) microarray analysis was carried out followed by whole exome sequencing of the affected proband and another affected sibling. Shared homozygous regions in all severely affected members were identified through homozygosity mapping approach.Results: The largest homozygous region of 14.1 Mb shared by the five severely affected members of the family was identified on chromosome 2. Subsequent exome sequencing identified a novel single nucleotide deletion c.2658del; p.(Ser887Profs*64) in KIF1A. Segregation analysis revealed that this mutation was homozygous in all five affected individuals of the family with severe clinical manifestation, while members of the family that were heterozygous carriers shared abnormal skin features (scaly skin) only with the homozygous affected members.Conclusions: A novel frameshift mutation p.(Ser887Profs*64) in KIF1A is the potential cause of severe HSANIIC in a Pakistani family along with incomplete penetrance in mutation carriers. We demonstrate that using a combination of different techniques not only strengthens the gene finding approach but also helps in proper sub-clinical characterization along with identification of mutated alleles exhibiting incomplete penetrance leading to intrafamilial clinical variability in HSAN group of inherited diseases.

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle.

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

Pseudorabies Virus Infection Accelerates Degradation of the Kinesin-3 Motor KIF1A.

Alphaherpesviruses, including pseudorabies virus (PRV), are neuroinvasive pathogens that establish lifelong latency in peripheral ganglia following the initial infection at mucosal surfaces. The establishment of latent infection and subsequent reactivations, during which newly assembled virions are sorted into and transported anterogradely inside axons to the initial mucosal site of infection, rely on axonal bidirectional transport mediated by microtubule-based motors. Previous studies using cultured peripheral nervous system (PNS) neurons have demonstrated that KIF1A, a kinesin-3 motor, mediates the efficient axonal sorting and transport of newly assembled PRV virions. Here we report that KIF1A, unlike other axonal kinesins, is an intrinsically unstable protein prone to proteasomal degradation. Interestingly, PRV infection of neuronal cells leads not only to a nonspecific depletion of KIF1A mRNA but also to an accelerated proteasomal degradation of KIF1A proteins, leading to a near depletion of KIF1A protein late in infection. Using a series of PRV mutants deficient in axonal sorting and anterograde spread, we identified the PRV US9/gE/gI protein complex as a viral factor facilitating the proteasomal degradation of KIF1A proteins. Moreover, by using compartmented neuronal cultures that fluidically and physically separate axons from cell bodies, we found that the proteasomal degradation of KIF1A occurs in axons during infection. We propose that the PRV anterograde sorting complex, gE/gI/US9, recruits KIF1A to viral transport vesicles for axonal sorting and transport and eventually accelerates the proteasomal degradation of KIF1A in axons.IMPORTANCE Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens herpes simplex viruses 1 and 2 and varicella-zoster virus. Alphaherpesviruses are neuroinvasive pathogens that establish lifelong latent infections in the host peripheral nervous system (PNS). Following reactivation from latency, infection spreads from the PNS back via axons to the peripheral mucosal tissues, a process mediated by kinesin motors. Here, we unveil and characterize the underlying mechanisms for a PRV-induced, accelerated degradation of KIF1A, a kinesin-3 motor promoting the sorting and transport of PRV virions in axons. We show that PRV infection disrupts the synthesis of KIF1A and simultaneously promotes the degradation of intrinsically unstable KIF1A proteins by proteasomes in axons. Our work implies that the timing of motor reduction after reactivation would be critical because progeny particles would have a limited time window for sorting into and transport in axons for further host-to-host spread.

Phenotypic expansion in KIF1A-related dominant disorders: A description of novel variants and review of published cases.

KIF1A is a molecular motor for membrane-bound cargo important to the development and survival of sensory neurons. KIF1A dysfunction has been associated with several Mendelian disorders with a spectrum of overlapping phenotypes, ranging from spastic paraplegia to intellectual disability. We present a novel pathogenic in-frame deletion in the KIF1A molecular motor domain inherited by two affected siblings from an unaffected mother with apparent germline mosaicism. We identified eight additional cases with heterozygous, pathogenic KIF1A variants ascertained from a local data lake. Our data provide evidence for the expansion of KIF1A-associated phenotypes to include hip subluxation and dystonia as well as phenotypes observed in only a single case: gelastic cataplexy, coxa valga, and double collecting system. We review the literature and suggest that KIF1A dysfunction is better understood as a single neuromuscular disorder with variable involvement of other organ systems than a set of discrete disorders converging at a single locus.

Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A).

Defects in the motor domain of kinesin family member 1A (KIF1A), a neuron-specific ATP-dependent anterograde axonal transporter of synaptic cargo, are well-recognized to cause a spectrum of neurological conditions, commonly known as KIF1A-associated neurological disorders (KAND). Here, we report one mutation-negative female with classic Rett syndrome (RTT) harboring a de novo heterozygous novel variant [NP_001230937.1:p.(Asp248Glu)] in the highly conserved motor domain of KIF1A. In addition, three individuals with severe neurodevelopmental disorder along with clinical features overlapping with KAND are also reported carrying de novo heterozygous novel [NP_001230937.1:p.(Cys92Arg) and p.(Pro305Leu)] or previously reported [NP_001230937.1:p.(Thr99Met)] variants in KIF1A. In silico tools predicted these variants to be likely pathogenic, and 3D molecular modeling predicted defective ATP hydrolysis and/or microtubule binding. Using the neurite tip accumulation assay, we demonstrated that all novel KIF1A variants significantly reduced the ability of the motor domain of KIF1A to accumulate along the neurite lengths of differentiated SH-SY5Y cells. In vitro microtubule gliding assays showed significantly reduced velocities for the variant p.(Asp248Glu) and reduced microtubule binding for the p.(Cys92Arg) and p.(Pro305Leu) variants, suggesting a decreased ability of KIF1A to move along microtubules. Thus, this study further expanded the phenotypic characteristics of KAND individuals with pathogenic variants in the KIF1A motor domain to include clinical features commonly seen in RTT individuals.

Polyglutamylation of tubulin's C-terminal tail controls pausing and motility of kinesin-3 family member KIF1A.

The kinesin-3 family member KIF1A plays a critical role in site-specific neuronal cargo delivery during axonal transport. KIF1A cargo is mislocalized in many neurodegenerative diseases, indicating that KIF1A's highly efficient, superprocessive motility along axonal microtubules needs to be tightly regulated. One potential regulatory mechanism may be through posttranslational modifications (PTMs) of axonal microtubules. These PTMs often occur on the C-terminal tails of the microtubule tracks, act as molecular "traffic signals" helping to direct kinesin motor cargo delivery, and include C-terminal tail polyglutamylation important for KIF1A cargo transport. KIF1A initially interacts with microtubule C-terminal tails through its K-loop, a positively charged surface loop of the KIF1A motor domain. However, the role of the K-loop in KIF1A motility and response to perturbations in C-terminal tail polyglutamylation is underexplored. Using single-molecule imaging, we present evidence that KIF1A pauses on different microtubule lattice structures, linking multiple processive segments together and contributing to KIF1A's characteristic superprocessive run length. Furthermore, modifications of the KIF1A K-loop or tubulin C-terminal tail polyglutamylation reduced KIF1A pausing and overall run length. These results suggest a new mechanism to regulate KIF1A motility via pauses mediated by K-loop/polyglutamylated C-terminal tail interactions, providing further insight into KIF1A's role in axonal transport.

Exploring the possibility of predicting human head hair greying from DNA using whole-exome and targeted NGS data.

BACKGROUND: Greying of the hair is an obvious sign of human aging. In addition to age, sex- and ancestry-specific patterns of hair greying are also observed and the progression of greying may be affected by environmental factors. However, little is known about the genetic control of this process. This study aimed to assess the potential of genetic data to predict hair greying in a population of nearly 1000 individuals from Poland. RESULTS: The study involved whole-exome sequencing followed by targeted analysis of 378 exome-wide and literature-based selected SNPs. For the selection of predictors, the minimum redundancy maximum relevance (mRMRe) method was used, and then two prediction models were developed. The models included age, sex and 13 unique SNPs. Two SNPs of the highest mRMRe score included whole-exome identified KIF1A rs59733750 and previously linked with hair loss FGF5 rs7680591. The model for greying vs. no greying prediction achieved accuracy of cross-validated AUC = 0.873. In the 3-grade classification cross-validated AUC equalled 0.864 for no greying, 0.791 for mild greying and 0.875 for severe greying. Although these values present fairly accurate prediction, most of the prediction information was brought by age alone. Genetic variants explained < 10% of hair greying variation and the impact of particular SNPs on prediction accuracy was found to be small. CONCLUSIONS: The rate of changes in human progressive traits shows inter-individual variation, therefore they are perceived as biomarkers of the biological age of the organism. The knowledge on the mechanisms underlying phenotypic aging can be of special interest to the medicine, cosmetics industry and forensics. Our study improves the knowledge on the genetics underlying hair greying processes, presents prototype models for prediction and proves hair greying being genetically a very complex trait. Finally, we propose a four-step approach based on genetic and epigenetic data analysis allowing for i) sex determination; ii) genetic ancestry inference; iii) greying-associated SNPs assignment and iv) epigenetic age estimation, all needed for a final prediction of greying.

KIF1A-related autosomal dominant spastic paraplegias (SPG30) in Russian families.

BACKGROUND: Spastic paraplegia type 30 (SPG30) caused by KIF1A mutations was first reported in 2011 and was initially considered a very rare autosomal recessive (AR) form. In the last years, thanks to the development of massive parallel sequencing, SPG30 proved to be a rather common autosomal dominant (AD) form of familial or sporadic spastic paraplegia (SPG),, with a wide range of phenotypes: pure and complicated. The aim of our study is to detect AD SPG30 cases and to examine their molecular and clinical characteristics for the first time in the Russian population. METHODS: Clinical, genealogical and molecular methods were used. Molecular methods included massive parallel sequencing (MPS) of custom panel 'spastic paraplegias' with 62 target genes complemented by familial Sanger sequencing. One case was detected by the whole -exome sequencing. RESULTS: AD SPG30 was detected in 10 unrelated families, making it the 3rd (8.4%) most common SPG form in the cohort of 118 families. No AR SPG30 cases were detected. In total, 9 heterozygous KIF1A mutations were detected, with 4 novel and 5 known mutations. All the mutations were located within KIF1A motor domain. Six cases had pure phenotypes, of which 5 were familial, where 2 familial cases demonstrated incomplete penetrance, early onset and slow relatively benign SPG course. All 4 complicated cases were caused by novel mutations without familial history. The phenotypes varied from severe in two patients (e.g. lack of walking, pronounced mental retardation) to relatively mild non-disabling symptoms in two others. CONCLUSION: AD SPG30 is one of the most common forms of SPG in Russia, the disorder has pronounced clinical variability while pure familial cases represent a significant part.