Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced β-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3′ UTR of β-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with β-actin mRNA and translocates to axons and growth cones of motoneurons.

This cross-sectional cohort study provides the first large-scale data on disease manifestation, progression, and modifying factors, with relevance for counseling of HSP families and planning of future cross-sectional and natural history studies. Later age of onset, specific complicating features, and the SPG11 genotype are strongly associated with more severe disease. Future interventional studies will require stratification for modifiers of disease progression identified in this study. Prospective longitudinal studies will verify progression rates calculated in this baseline analysis.

BackgroundMutations in SACS, leading to autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), have been identified as a frequent cause of recessive early-onset ataxia around the world. Here we aimed to enlarge the spectrum of SACS mutations outside Quebec, to establish the pathogenicity of novel variants, and to expand the clinical and imaging phenotype.MethodsSequencing of SACS in 22 patients with unexplained early-onset ataxia, assessment of novel SACS variants in 3.500 European control chromosomes and extensive phenotypic investigations of all SACS carriers.ResultsWe identified 11 index patients harbouring 17 novel SACS variants. 9/11 patients harboured two variants of at least probable pathogenicity which were not observed in controls and, in case of missense mutations, were located in highly conserved domains. These 9 patients accounted for at least 11% (9/83) in our series of unexplained early onset ataxia subjects. While most patients (7/9) showed the classical ARSACS triad, the presenting phenotype reached from pure neuropathy (leading to the initial diagnosis of Charcot-Marie-Tooth disease) in one subject to the absence of any signs of neuropathy in another. In contrast to its name “spastic ataxia”, neither spasticity (absent in 2/9=22%) nor extensor plantar response (absent in 3/9=33%) nor cerebellar ataxia (absent in 1/9=11%) were obligate features. Autonomic features included urine urge incontinence and erectile dysfunction. Apart from the well-established MRI finding of pontine hypointensities, all patients (100%) showed hyperintensities of the lateral pons merging into the (thickened) middle cerebellar peduncles. In addition, 63% exhibited bilateral parietal cerebral atrophy, and 63% a short circumscribed thinning of the posterior midbody of the corpus callosum. In 2 further patients with differences in important clinical features, VUS class 3 variants (c.1373C>T [p.Thr458Ile] and c.2983 G>T [p.Val995Phe]) were identified. These variants were, however, also observed in controls, thus questioning their pathogenic relevance.ConclusionsWe here demonstrate that each feature of the classical ARSACS triad (cerebellar ataxia, spasticity and peripheral neuropathy) might be missing in ARSACS. Nevertheless, characteristic MRI features – which also extend to supratentorial regions and involve the cerebral cortex – will help to establish the diagnosis in most cases.

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.

Axonopathies are a group of clinically diverse disorders characterized by the progressive degeneration of the axons of specific neurons. In hereditary spastic paraplegia (HSP), the axons of cortical motor neurons degenerate and cause a spastic movement disorder. HSP is linked to mutations in several loci known collectively as the spastic paraplegia genes (SPGs). We identified a heterozygous receptor accessory protein 1 (REEP1) exon 2 deletion in a patient suffering from the autosomal dominantly inherited HSP variant SPG31. We generated the corresponding mouse model to study the underlying cellular pathology. Mice with heterozygous deletion of exon 2 in Reep1 displayed a gait disorder closely resembling SPG31 in humans. Homozygous exon 2 deletion resulted in the complete loss of REEP1 and a more severe phenotype with earlier onset. At the molecular level, we demonstrated that REEP1 is a neuron-specific, membrane-binding, and membrane curvature-inducing protein that resides in the ER. We further show that Reep1 expression was prominent in cortical motor neurons. In REEP1-deficient mice, these neurons showed reduced complexity of the peripheral ER upon ultrastructural analysis. Our study connects proper neuronal ER architecture to long-term axon survival.

Motor neuron degeneration is the predominant pathological feature of spinal muscular atrophy (SMA). In patients with severe forms of the disease, additional sensory abnormalities have been reported. However, it is not clear whether the loss of sensory neurons is a common feature in severe forms of the disease, how many neurons are lost and how loss of sensory neurons compares with motor neuron degeneration. We have analysed dorsal root ganglionic sensory neurons in Smn-/-;SMN2 mice, a model of type I SMA. In contrast to lumbar motor neurons, no loss of sensory neurons in the L5 dorsal root ganglia is found at post-natal days 3-5 when these mice are severely paralyzed and die from motor defects. Survival of cultured sensory neurons in the presence of NGF and other neurotrophic factors is not reduced in comparison to wild-type controls. However, isolated sensory neurons have shorter neurites and smaller growth cones, and beta-actin protein and beta-actin mRNA are reduced in sensory neurite terminals. In footpads of Smn-deficient mouse embryos, sensory nerve terminals are smaller, suggesting that Smn deficiency reduces neurite outgrowth during embryogenesis. These data indicate that pathological alterations in severe forms of SMA are not restricted to motor neurons, but the defects in the sensory neurons are milder than those in the motor neurons.

Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.

Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder preferentially affecting the longest corticospinal axons. More than 40 HSP genetic loci have been identified, among them SPG10, an autosomal dominant HSP caused by point mutations in the neuronal kinesin heavy chain protein KIF5A. Constitutive KIF5A knockout (KIF5A–/–) mice die early after birth. In these mice, lungs were unexpanded, and cell bodies of lower motor neurons in the spinal cord swollen, but the pathomechanism remained unclear. To gain insights into the pathophysiology, we characterized survival, outgrowth, and function in primary motor and sensory neuron cultures from KIF5A–/– mice. Absence of KIF5A reduced survival in motor neurons, but not in sensory neurons. Outgrowth of axons and dendrites was remarkably diminished in KIF5A–/– motor neurons. The number of axonal branches was reduced, whereas the number of dendrites was not altered. In KIF5A–/– sensory neurons, neurite outgrowth was decreased but the number of neurites remained unchanged. In motor neurons maximum and average velocity of mitochondrial transport was reduced both in anterograde and retrograde direction. Our results point out a role of KIF5A in process outgrowth and axonal transport of mitochondria, affecting motor neurons more severely than sensory neurons. This gives pathophysiological insights into KIF5A associated HSP, and matches the clinical findings of predominant degeneration of the longest axons of the corticospinal tract.Electronic supplementary materialThe online version of this article (doi:10.1007/s10048-012-0324-y) contains supplementary material, which is available to authorized users.