Hereditary Spastic Paraplegias. Clinical Spectrum and Growing List of Genes

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1 SMGr up Hereditary Spastic Paraplegias. Clinical Spectrum and Growing List of Genes Alice K Abdel Aleem, MB, B.Ch., MD Department of Neurology, Weill Cornell Medical College Qatar, Qatar *Corresponding author: Alice Abdel Aleem, Department of Neurology, Neurogenetics program, Research Division, Weill Cornell Medical College Qatar, Education City, Doha, Qatar, aka2005@qatar-med.cornell.edu This work was funded by Qatar Foundation National Priority Fund, Grant NPRP Published Date: March 11, 2017 ABSTRACT Hereditary spastic paraplegias (HSPs) constitute a large growing group of genetically determined neurodegenerative disorders. The basic pathology in HSPs is degeneration of axons of the corticospinal tract, the long tract connecting cerebral cortical neurons to spinal cord ones. Axonal degenerative process is length dependent and starting most distally, the reason for the given designation of distal axonopathy or retrograde degeneration. Typically, HSPs are characterized by lower limbs progressive spasticity, muscle weakness, pyramidal signs of brisk deep tendon reflex, hypertonia, and extensor planter response, as well as associated urinary sphincter disturbance. HSPs group of disorders are one of the most clinically and genetically heterogeneous human genetic disorders. The several molecular complex mechanisms involved in axonal transport, membrane and organelle trafficking, organelle morphogenesis and distribution, 1

2 maintenance of healthy environments of corticospinal tract s axons, as well as the lipid/ sphingolipid/phosphatases/ and nucleotide metabolisms are the basics for the extensive genetic heterogeneity and the evolving list of genes that are expanding and discovered in relation to HSPs. This genetic heterogeneity is projected on the occurrence of various inheritance patterns; autosomal recessive, dominant, X-linked, sporadic, or mitochondrial and the association of a diverse of clinical presentations, as well as the notable phenotypic overlaps. Clinical heterogeneity in HSPs displayed marked intra- and inter-familial variability in disease age of onset, presentations spectrum, progression severity, and importantly is the association of severe neurodevelopmental, cerebellar ataxia, lower motor neuron, or neuropathy signs. In the light of such genotype and phenotype complexity, whole exome sequencing (WES) or whole genome sequencing (WGS) has progressively increasing the ability of identification of causative genes. Clinical WES is currently evolved into a mandatory diagnostic genetic tool to uncover the genetic defects in familial or sporadic HSPs. Keywords: Hereditary spastic paraplegia; HSPs; Clinical and genetic heterogeneity; Axonal degeneration; Corticospinal tract degenration; HSPs molecular mechanisms; Spastic gait differential diagnosis HSP OVERVIEW Hereditary spastic paraplegias (HSPs) or spastic paraplegia (SPGs) describes a huge, extensively heterogeneous group of inherited neurodegenerative disorders characterized mainly by spasticity and weakness (Paraparesis) of the lower limbs [1,2]. Inheritance of HSPs involves the three modes of Mendelian inheritance; Autosomal dominant, Autosomal recessive and X-linked, in addition to the non-mendelian mitochondrial transmission [2-6]. There are several molecular and pathophysiological mechanisms described as HSPs causal [7-10]. However, the main pathology underlies the typical HSPs disease features of lower limb spasticity and muscle weakness is the retrograde degeneration of the descending motor fibers axons of the corticospinal tract and degeneration of the posterior column of the spinal cord [9,11,12]. Corticospinal tract is the main motor pathway controlling voluntary movements, connecting upper motor neuronal cells in cerebral cortex grey matter to lower motor neuronal cells in spinal cord grey matter. Axons of the lower motor neurons (spinal cord grey matter neuronal cells) synapse with the motor end plate at the neuromuscular junctions mediating motor voluntary movements. HSPs is a kind of upper motor neuron involvements, however, the upper motor neuron axonopathy is a rather specific designation. In HSPs, axonal degeneration started at the most distal ends and progress further cranially toward the neuronal cells. Interestingly, there is little neuronal death in HSPs. High throughput sequencing tools, WES and WGS enabled the identification of an expanding list of genes contributing to SPGs causality [13,14]. Investigators efforts were, to a great extent, successful in gathering or relating different SPGs genes, to intersecting functional pathways or to particular molecular 2

3 mechanisms. Membrane trafficking/axonal transport related cell biological process involves the ER and Endosomal morohogenesis, ER membrane proteins, Endosomal traffic, ER-Golgi traffic, Cytoskeletal (microtubules and motor molecules) regulation, Lipid droplet biogenesis, and mitochondrial related functions [2,8,9,11,15]. Such complex machineries function in establishing a proper intracellular sorting, distribution, and transport of proteins and organelles, anterograde and retrograde, along the long corticospinal tract s axons as well as proper maintenance of axonal myelination. Understanding these mechanisms contributing to either axonal maintenance or degeneration is of a great clinical relevance to better understanding of the rather common neurological disorders mainly those due to axonopathies, like neuropathies or multiple sclerosis. Up to date, more than 70 Spastic paraplegias (SPGs) with identified genes or assigned loci were described encompassing 19 AD-SPGs, 48 AR SPGs, 5 X-linked SPGs,4 mitochondrial genome associated SPGs (maternal inheritance) (Table 1). HSPs overall prevalence was estimated, in an updated study, around 1.8/ Other studies estimated a range of HSPs prevalence between in [16,17]. HSPS PHENOTYPES Typical HSPs Presentations There are standard SPG s characteristics with or without variable association of neurological and non-neurological symptoms and signs. The main clinical features that points out a case with a provisional clinical diagnosis of HSP involve: (i) bilateral lower limbs spasticity and weakness with repeated falling and tip-toe walking or spastic gait; (ii) signs of pyramidal involvements; exaggerated/brisk deep tendon reflexes (DTRs) with ankle clonus in some cases, spasticity in the form of limited ankle dorsiflexion, limited hips abduction, and up-going planter reflex positive Babinski ; (iii) upper limbs are spared with normal tone and DTRs, in almost most of the cases; (iv) urinary urgency is of the HSPs primary presentation, however not in all cases; (v) familial or sporadic; the proband can be the first case in the family or has multiple affected family members with or without remote positive family history. HSPs Clinical Classification {Clinical heterogeneity} HSPs diagnosis should be primarily preceded by imaging of spines, spinal cord and brain to exclude structural causes of myelopathies or cerebral palsy. HSPs is known to be largely clinically heterogeneous with a wide spectrum of presentations ranging from pure HSP of only the standard features of LL spasticity and weakness to HSP s features associated with mental and cognitive involvements, ataxia, optic atrophy, and brain imaging abnormalities among other features [8,18,19]. 3

4 Pure forms of HSPs are described in patients who presented with rather a pure presentation of progressive pyramidal signs [brisk DTR, spasticity, hypertonia, and positive Babinski] and symptoms [tip toe gait, delayed crawling and/or walking, lower limbs weakness and frequent falling] with the common association of urinary bladder urgency. Motor development, muscle power, cognition, speech, and sensation are described as normal in pure forms of HSPs. Pure HSPs is the predominant presentation among most of the AD-HSPs, whereas it is not frequently encountered in AR forms. Complex HSPs (Table 1) characterized by the combined presentations of typical HSP pyramidal signs and symptoms together with the variable association of other neurologic and non-neurologic features. Associated neurological manifestations in complex forms involve cerebellar ataxia, nystagmus, a spectrum of cognitive impairments [ranging from intellectual disability to variable degrees of mental retardation up to dementia], delay in developmental milestones including speech delay and dysarthria, peripheral neuropathy, myopathic changes including muscles of the eye [ptosis and external ophthalmoplegias], convulsions, extrapyramidal manifestations [dystonia, involuntary movements, tremors or parkinsonism], brain imaging abnormalities [leukodytrophy, cerebellar hypoplasia/atrophy, small brain stem, thin/dysgenic/hypoplastic corpus callosum, brain iron accumulation, or hydrocephalus]. Other manifestations involved strabismus, optic atrophy, macular degeneration, icthyosis, facial dysmorphism, micro- or macro-cephaly, adducted thumb, Talebus deformities, congenital hip dislocation, or kyphosis/scoliosis/kyphoscoliosis. Pure HSPs phenotype is more commonly seen in families with AD-HSPs. Pure autosomal dominant HSPs described as the prevalent forms in specific population of Northern Europe, North America, and Japan [20-22]. Complicated HSPs phenotypes are mostly inherited as Autosomal Recessive group of disorders [10,23], which signifies the growing list of newly discovered HSP-related genes. AR-HSPs are more prevalent in Arab, Mediterranean, and non-european countries, the population with high rate of consanguineous marriages. The prevalence rate was estimated in Tunisia as 5.75/ versus 0.6/ in Norway [24,25]. Neuroimaging Features in HSPs Phenotypes Brain MRI imaging in patients with HSPs are variable and mostly nonspecific describing white matter (WM) lesions or signal intensities abnormalities involving high signal intensity of the posterior limb of the internal capsule in T2 images, where the corticospinal tracts are routing in their way to the brain stem and spinal cord [26]. Interesting brain MRI studies in HSPs patients, involving a quantitative MRI of brain volume reported a notable brain atrophy of both the grey and white matters in complex HSPs patients compared to age matched controls. Studies applied single-photon emission CT (SPECT) in HSPs revealed progressively metabolism of thalamus and cerebral cortex; frontal, temporal 4

5 and parietal regions [26-29]. Several other studies reported a significant atrophy of the spinal cord at the cervical and thoracic levels in AD and AR HSPs patients compared to controls [30-32]. Thin Corpus Callosum (TCC), Brain Iron Accumulation (BIA), cerebellar hypoplasia/atrophy (CH/CA) are of the particulars seen in brain imaging of patients with certain HSPs subgroups. TCC in HSPs patients is documented to be frequently accompanied with the presentation of intellectual disability or cognitive involvements [33,34]. SPG11 and SPG 15 are the most common ARHSPs found to be associated with TCC [35,36], however; TCC was variably reported in other subtypes that were collectively labeled as HSPs with TCC subgroup. Neurodegeneration with Brain Iron Accumulation (NBIA), particularly in basal ganglion,is a group of rare autosomal recessive neurodegenerative diseases that involves a spectrum of distinguished clinical disorders of which SPG35 with mutations in FA2H has been reported [37]. Association of cerebellar atrophy (CA) or hypoplasia (CH) in brain imaging of HSPs is commonly associated with clinical presentation of cerebellar signs [7] involving dysarthric speech (the most common associated sign in patients with complex HSP), ataxia that can be a prominent feature or only an associated presentation, coordination involvements, and nystagmus. Rare subgroups of complex HSPs classified on the basis of particular neuroimaging characteristics (i) (ii) Thin Corpus Callosum SPG s subgroup (TCC-SPG) : SPGs with a notably thin or dysgenic corpus callosum seen in brain imaging of patients, mostly with complex forms of AR-HSP. (Figure 1 and Figure 2). TCC-SPG subgroup involves SPG11 (SPG11) [33], SPG15 (ZFYVE26) [35], SPG 7 (SpG7) [38,39], SPG21 (ACP33) [40], SPGs 44 (GJC2) [41], SPG 46 (GBA2) [42], SPG 47 (AP4B1) [43], SPG56 (CYP2U1) [44], SPG 54(DDHD2) [45], SPG45/65(NT5C2) [13]. Brain iron accumulation SPG s subgroup (NBIA-SPG) : SPG characterized by the association of progressive extra pyramidal and/or parkinsonian manifestations (dystonia, choreoathetosis, rigidity) with brain imaging of iron accumulation, predominantly in the basal ganglia (figure 2). SPG35 with mutations in FA2H falls under this rare group of NBIA [46]. (iii) Cerebellar Hypoplasia/Cerebellar Atrophy, CH/CA is a brain imaging finding seen in a number of complex HSPs, in SPG46 (GBA2) [42,47] and SPG58 (KIF1C) [13,48], in particular, ataxia is of the prominent phenotypic presentations. Meanwhile, the CH in brain imaging of other SPGs forms seems rather as an associated sign [7,8]: SPG1 (L1CAM), SPG7 (SPG7), SPG11 (SPG11), SPG15 (ZFYVE26), SPG26 (B4GALNT1), SPG30 (KIF1A), SPG31 (REEP1), SPG44 (GJC2), SPG49/SPG56 (CYP2U1), SPG50 (AP4M1), SPG54 (DDHD2), SPG66 (ARSI), and SPG67 (PGAP1). 5

6 Figure 1: Brain MRI scan of two identical Egyptian twins with SPG11 from Abdel Aleem et al. [33]. Images A and B, feature TCC in both twins (black arrows), C and F, white matter hyper intensities (white arrow head). Cerebellar arachnoid cyst was present only in one of the twin, image E (large black arrow). Figure 2: Brain MRI of a case with FA2H mutation from Krueret al. [46]. Fig. 2A: Shows hypointense black signal in the globus Palidus [basal ganglion] consistent with iron accumulation [blue arrow head]. Fig 2B: Shows white matter hyper intensities. Fig 2C: Reveals TCC [pink arrow] and brain stem, spinal cord, and cerebellar atrophy (pontocerebellar atrophy) [yellow arrows]. 6

7 Table 1: Inheritance, genetics, and phenotypic features of hereditary spastic paraplegias (HSPs). Genetic Inheritance SPG subtype/ chr. number/ gene name Protein AD SPG3A /14q/ ATL1 Atlastin1. Dynamin related GTPase/ Golgi transmembrane protein Putative protein function/molecular mechanisms Neurite outgrowth, interact with Spastin functions in membrane trafficking, ER& Golgi morphology and trafficking, BMP signaling SPG4/2p/ SPAST Spastin Microtubule disassembly & dynamics, axonal transport, membrane trafficking, ER morphogenesis, BMP signaling SPG6/ 15q/ NIPA1 [nonimprinted gene in Prader- Willi/Angelman chromosome region] SPG8/ 8q/ KIAA0196 SPG9/10q/ ALDH18A1 [Aldehyde dehydrogenase18 A1] SPG10/12q/ KIF5A NIPA neural protein Strumpellin Glutamate semialdehyde synthetase Kinesin heavy chain 5A. Endosomal/ER morphogenesis, protein folding, magnesium ion metabolism in the cell., BMP signaling Ubiquitously expressed in cytoplasm and ER. Endosomal morphogenesis, protein folding Denovo biosynthesis of Ornithine, proline, & arginine. Axonal transport anterograde-microtubule related motor protein molecules transport Clinical phenotype Main typical presentation is pure HSP with urinary sphincter disturbance. Variable age of onset and disease progression. Atypical complex presentation of mild ID, TCC (present or absent) Typical presentation of Pure HSP, variability in severity and age of onset. Atypical complex cases with nystagmus, ID, behavioral abnormalities, neuroimaging abnormalities. Pure and complex [seizures, ID, neuropathies] HSP s phenotypes. Phenotypes of pure (with severe spasticity) and complex [dysphagia] HSP SPG9A, a dominant complicated HSP, slowly progressive, associated with dysarthria, motor neuropathy, gastro-esophageal reflux (vomiting), bones dysplasia [dysplastic hips, carpal bones], cataract, short stature, amyotrophy. SPG9B, a recessive, complicated HSP seen in Spanish and Portuguese families with sever ID, psychomotor retardation, microcephaly, dysmorphic, generalized amyotrophy. Pure and complex [parkinsonism, ID, ataxia, PN, distal amyotrophy, scoliosis. SPG12/ 19q/ Reticulon2 ER morphogenesis, A typical pure HSP of variable 61, Ref 8, 49, 50 15, 51, 52 53, 54 3, 55, 56 7,8, 57 3, 58, 59, 60 7

8 RTN2 interacts with spastin age of onset 62 SPG13/ 2q/ HSPD1/ HSPD60 Chaperonin [mtheat shock 60KD protein 1 Protein folding and assembly in mitochondria Pure HSP with severe spasticity 7, 63, 64 SPG17/ 11q/ BSCL2 Silver syndrome SPG19/9q/ {locus} SPG29/1p/ {locus} SPG31/ 2p/ REEP1 SPG33/10q / ZFYVE27 SPG36/ 12q {locus} Seipin ER protein Lipid metabolism, adipogenesis, ER stress response HSP associated with distal lower limb amyotrophy, wasting of hand muscles [then arms & dorsal interossei] - - Typical HSP associated with motor neuropathy - - HSP associated with hiatal & esophageal hernia, hearing loss Receptor expression enhancing protein1 ER morphogenesis, mitochondrial chaperon like activity Pure HSP, association with distal amyotrophy, &/or dysphagia was reported. Rarely, few patients showed complicated forms. Protrudin Spastin binding protein Pure HSP Pure HSP, association with PN , SPG37/ 8P {locus} - - Pure HSP with urinary sphincter disturbance, slowly progressive course and variable age of onset SPG38 /4P {locus} SPG41 /11P {locus} SPG42/3q/ SLC33A1 - - Pure HSP associated with distal amyotrophy, lower motor neuron phenotype, similar to HSP Pure HSP, urinary sphincter disturbance, slowly progressive course. Acetyl-coenzyme A transporter1 in Golgi Transport acetyl-coa into lumen of Golgi apparatus Pure HSP, variable age of onset, slowly progressive course, pes cavus SPG73/19q/ CPT1C Carnitine palmitoyl Neuronal isoform localized to ER, not to mitochondria, Reported in a large Italian family as adult onset slowly 80 8

9 (Carnitine Palmitoyl Transferase 1C) transferase in the soma, dendrites, and projections of axons of motor neuron. Little activity in B-oxidation of long chain fatty acids (LCFA) compared to CPT1A & B that transport LCFA from cytoplasm to mitochondrial matrix. CPT1C Interacts with atlastin1. A role in altered lipid mediated signal transduction. progressive pure HSP associated with mild proximal muscle wasting and atrophy, urinary dysfunction, feet deformities. AR SPG5A/8q/ CYP7B1 [Cytochrome P450 Family7, subfamily B1] SPG7/16q/ SPG7 (PNG) {rarely of AD or sporadic inheritance} SPG11/15q/ SPG11 25-hydroxycholesterol 7- alphahydroxylase Paraplegin Spatacsin Steroid/lipid metabolism, generation of neuroprotective steroids Mitochondrial protease Involved in degradation of misfolded proteins and regulation of ribosomes association at the inner mitochondrial membrane ATP-proteolytic complex. Neuronal growth, vesicles sorting and transport, intracellular cargo trafficking. Pure HSP forms of variable age of onset & slow progression course. Complex forms with spastic, cerebellar ataxia, optic atrophy. NI: cerebellar &spinal cord atrophy, WM changes. Phenotypes either of rather pure forms, however with dysarthria, pes cavus, cerebellar ataxia or complex forms with optic atrophy, ID, ophthalmoparesis, nystagmus, scoliosis. NI: cerebral and cerebellar atrophy with WM lesions. SPG7 causative in sporadic or AR pure cerebellar ataxia. Typical HSP associated with cerebellar ataxia, ID, PN, distal amyotrophy, dysphagia, parkinsonism, macular degeneration over years. NI: TCC, mild VD, periventricular WM changes, cortical atrophy, sign of ears of the lynx. SPG14/3q/{locus} - - Complex form of HSP with slowly progressive course, ID, distal motor neuropathy SPG15/ 14q / ZFYVE26 Spastizin Spinal motor neuronal axons outgrowth, lysosomal tubulation, lysosomal autophagic Complex slowly progressive HSP phenotype with cerebellar ataxia, distal amyotrophy, demyelinating and axonal 3, 81, 82 39, , 85, 33, , 36, 88, 89 9

10 reformation. Secretory vesicles maturation, endosomal trafficking, cytokinesis SPG18/8p/ ERLIN Erlin2/SPFH2 ERAD pathway regulator. Modulate the ERassociated degradation pathway [ERAD] of inositol triphosphate receptors [IP3Rs] involved in intracellular cholesterol homeostasis. SPG20/13q/ SPG20 [Troyer syndrome] SPG21/15q/ SPG21 or ACP33 [Acidic Cluster Protein 33KD] Mast syndrome SPG23/1q DSTYK (RIP5) Spartin Co-localize with mitochondria, partially colocalize with microtubule Maspardin DSTYK [Dusty protein kinase] or RIP5 [receptor interacting protein 5] Involved in lipid droplets turn over. Intracellular trafficking of epidermal growth factor receptor [EGFR], endosomal trafficking, mitochondrial function, microtubule dynamics. Endocytoplasmic trafficking, vesicles sorting and protein transport in trans-golgi. Dual serine-threonine and tyrosine protein kinase. A role in regulating cell death. polyneuropathy, seizures, pes cavus, hearing loss, variable degrees of mental involvements, retinal degeneration [reduced visual acuity] sometimes macular degeneration, psychosis, parkinsonism. NI: cortical atrophy, TCC, WM changes Severe progressive complex HSP with marked psychomotor and mental retardation, seizures, multiple joints contractures, scoliosis. Childhood onset HSP with distal, hands and feet muscle wasting, kyphoscoliosis, multiple joint contractures, hands joints hyperextensibility, clinodactyly, camptodacyly, pes cavus, developmental delay, cerebellar signs, dysmorphic features of hypertelorism & maxillary overgrowth, short stature. NI: cortical and cerebellar atrophy, WM changes. Slowly progressive complex HSP with extrapyramidal &cerebellar signs, bulbar dysfunction, PN, MR, severe dementia, NI: TCC, cortical frontotemporal atrophy HSP complex phenotype with progressive loss of skin and hair pigmentation [general or patchy dyspigmentation] Peripheral neuropathy/ epilepsy, mild MR, early graying of hair, urinary developmental defects, thin face, micrognathia & microcephaly. 90, 91 8, 92, 93 8,

11 SPG24/13q /{locus} - - Early onset pure HSP phenotype. SPG25/6q /{locus} - - Pure HSP with characteristic intervertebral cervical and lumber disk herniation, pain radiating to upper and lower limbs. SPG26/12q/ B4GALNT1 SPG27/10q/ {locus} Beta 1,4 N - Acetylgalactosaminyltransferase 1 Lipid metabolism/ ganglioside biosynthesis Slowly progressive, variable onset complex HSP with distal amyotrophy, PN, pes cavus, scoliosis, mild MR, extrapyramidal dystonia, & dyskinesais, cerebellar ataxia, nystagmus, cataracts - - Pure HSP phenotype SPG28/14q/ DDHD1 SPG30/2q /KIF1A SPG32/14q /{locus} SPG35/16q/ FA2H SPG39/19p/ PNPLA6 Phosphatidic acid PhospholipaseA1 [PAPLA1] Kinesin-like protein KIF1A Lipid/phospholipid/fatty acid metabolism. Maintenance of organelle ER/Golgi membrane and intracellular trafficking. Motor proteins, anterograde axonal transport of synaptic vesicles. Slowly progressive, variable age of onset HSP with scoliosis, pes cavus, cerebellar oculomotor disturbance, axonal PN. Brain & skeletal muscles reduced energy metabolism in magnetic resonance spectroscopy HSP with mild cerebellar ataxia & distal axonal neuropathy. NI: mild cerebellar atrophy. - - Slowly progressive complex HSP with mild MR. NI: TCC, cortical & cerebellar atrophy. Fatty acid 2 hydroxylase Neuropathy target esterase [NTE]. NTE-motor neuron disease Lipid /sphingolipid metabolism De-esterification of membrane phosphatidylcholine. Phospholipid homeostasis, motor neuron membrane integrity. Early onset complex HSP with mild MR, cerebellar ataxia, nystagmus, dysarthria, strabismus, optic atrophy, external ophthalmoplegia, dystonia. NI: TCC, periventricular WM hyperintensities, iron deposition in BG, mild cortical & pontocerebellar atrophy. Slowly progressive rather pure HSP with notable upper & lower limbs distal amyotrophy, axonal neuropathy. NI: cerebellar & spinal cord 44 8, 102 3, 7,

12 SPG43/19p/ C19ORF12 SPG44/1q/ GJC2 SPG45 / 10q/ NT5C2 [designated also as SPG65] SPG46/9p/ GBA2 SPG47/1p/ AP4B1 SPG48/7p/ AP-5Z1 SPG49/14q/ TECPR2 Protein C19ORF12, mitochondrial & ER protein. Gap junction gamma-2 Protein, connexin 47 protein. Nucleotidase cytosolic 2 Non-lysosomal Glucosylceramidase AP-4 complex subunit beta-1 AP-5 complex subunit zeta-1 Tectonin B- propeller repeatcontaining protein2. thoracic atrophy - HSP associated with dysarthria, marked muscle atrophy of limbs, disuse-joints contractures. Gap junctions formation, HSP with cerebellar ataxia, cell to cell interactions, dysarthria, pes cavus, lumber facilitate diffusion of ions lordosis, scoliosis, seizures, and small molecules hearing loss. NI: TCC, WM hypomyelination Cytoplasmic Hydrolase specific to IMP releasing adenosine. Functions in nucleotide purine metabolism Lipid/ganglioside metabolism Endosome Cargo transport, Vesicles formation and trafficking Endosomal transport interacting with spatacsin and spastizin, DNA (double stranded) repair helicase Intracellular lysosomal autophagy pathway Early onset slowly progressive complex HSP associated with mild ID, DD. NI: TCC and WM changes. Slowly progressive HSP, urinary incontinence, cerebellar ataxia, kyphoscoliosis, pes cavus, +/- MR, head tremor, dysarthria, dementia, congenital cataracts, hearing loss, small testicles with infertility. NI: TCC, cerebral & cerebellar atrophy Neonatal hypotonia, spastic paraplegia, MR, lack of speech, dysmorphisms (microcephaly, short philtrum, wide nasal bridge with bulbous nose, bitemporal narrowing, wide mouth), short stature, acetabular dysplasia, stereotypic movements, spastic tongue protrusion, dystonia, seizures. NI: TCC, WM changes, ventricular dilatation. HSP pure or complex with urinary incontinence, MR. NI: cervical spinal cord hyperintensities. Complex HSP with DD, ID, cerebellar ataxia, recurrent episodes of central apnoea, recurrent chest infections 2ry to gasteroesophageal reflux, dysmorphic features, microcephaly. NI: TCC, cerebral and cerebellar atrophy , , ,

13 SPG50/7q/ AP4M1 [cerebral palsy, spastic quadriplegic type 3] SPG51/15q/ AP4E1 cerebral palsy, spastic quadriplegic type 4] SPG52/ 14q/ AP4S1 Cerebral Palsy, Spastic quadriplegia type 6 SPG53/8p/ VPS37A SPG54/8p/ DDHD2 SPG55/ 12q/ C12ORF65 SPG56/4q/ CYP2U1 SPG57/3q/ TFG AP-4 complex subunit mu-1 AP-4 complex subunit epsilon 1 AP-4 complex subunit sigma 1 Vacuolar protein sorting 37 homolo.a Phospholipase DDHD2 Mitochondrial pr. Peptide release factor c12orf65 Cytochrome P450 2U1 TFG protein Cell Cargo transport, Vesicles formation and trafficking Cell Cargo transport, Vesicles formation and trafficking Cell Cargo transport, Vesicles formation and trafficking Vesicular traffecking and ubiquitination Lipid/phospholipid metabolism Mitochondrial peptide translational machinery Fatty acid hydroxylation, lipid/fatty acid metabolism vesicles formation and trafficking Neurodevelopmental phenotype of slowly progressive HSP associated with neonatal hypotonia evolved into spastic quadriplegia, lack of speech, severe MR, lack of sphincter control, strabismus, mild dysmorphic features, microcephaly. NI: TCC, cerebellar atrophy, WM changes, ventriculomegaly. Clinical and imaging picture is largely similar to SPG50 with additional findings of seizures, stereotypic laughter, nystagmus HSP clinical phenotype similar to SPG50 & SPG51 Complex HSP with developmental and speech delay, ID, kyphosis, sternum abnormalities, dystonia. Neurodevelopmental HSP phenotype with sychomotor and DD, ID, dysphagia, strabismus, optic nerve hypoplasia. NI: WM changes, TCC. Progressive complex HSP, progressive visual loss- related optic atrophy, psychomotor and MR, PN, arthrogryposis. Early onset HSP pure or complex forms with upper limbs dystonia, MR, axonal neuropathy subclinical. NI: TCC, WM changes. Early onset Complex HSP with early onset optic atrophy, axonal and demyelinating sensory-motor neuropathy. SPG58/17p/ Kinesin protein Retrograde motor transport Variable onset complex HSP ,

14 KIF1C 1C Golgi to ER with cerebellar ataxia, dysarthria, MR, microcephaly, hypodontia, extrapyramidal involuntary movements chorea. SPG59/15q/ USP8 SPG60/3p /WDR48 SPG61/16p/ ARL6IP1 SPG62/10q/ ERLIN1 SPG63/1p/ AMPD2 SPG64/10q/ ENTPD1 SPG66/5q/ ARSI SPG67/2q/ PGAP1 SPG68/11q/ FLRT1 SPG69/ 1q/ RAB3GAP2 Ubiquitin specific protease hydrolase 8 WDrepeatcontaining protein 48 ADP-ribosylationlike protein 6- interacting protein1 Erlin 1 [ER lipid RAFT associated 1] AMP deaminase 2 Ectonucleosidase triphosphate diphosphorilase 1 Arylsulphatase I Inositol deacylase Leucine-rich Transmembrane protein Rab3GTPaseactivating Protein 2-noncatalytic unit Maintain the morphology of the endosome by ubiquitination of its proteins & involved in the early stage of endosomal membrane trafficking. Protein regulator, regulator of deubiquitination Protein transport, membrane trafficking ER associated lipid degradation Purine nucleotide metabolism, AMP into IMP deamination Hydrolase regulating Purinergic transmission Hydrolysis of sulphates esters Inositol deactylation, nucleotide metabolism Cell adhesions and receptor signaling Non-catalytic GTPase activating protein with RAB3 specificity generates RAB3GDP required while brain and eye development. Rab3GAP2. Golgi-ER retrograde HSP with borderline intelligence. Infantile onset HSP with nystagmus, PN, ID. Complex HSP with severe sensory& motor polyneuropathy, loss of terminal digits severe acropathy. Complex HSP, cerebellar ataxia, amyotrophy Pure HSP, very slowly progressive, short stature. NI: WMC, TCC HSP slowly progressive, ID, microcephaly, delayed puberty, cerebellar signs, amyotrophy. NI: WM changes. Very early manifested HSP, severe sensory & motor polyneuropathy. NI: TCC, cerebellar hypoplasia HSP with global developmental delay, hand and feet deformities.ni: cortical atrophy, cerebellar hypoplasia, hypomyelination, CC agenesis. Typical HSP with mild spasticity, mild amyotrophy, nystagmus, optic atrophy. Complex HSP with Early onset spasticity, developmental delay, ID, dysarthria, cataracts, deafness ,

15 SPG70/ 12q/ MARS SPG71/5p/ ZFR SPG72/5q /REEP2 SPG74/1q/ IBA57 Methionine-tRNA ligase Zinc finger RNA binding protein Receptor expression enhancing protein2 IBA57 protein Iron-Sulfur cluster assembly mitochondrial protein transport, vesicles mediated transport, and Ca-dependent exocytosis of neurotransmitters is of the related pathways. Protein biosynthesis through its aminoacyl-trna activity RNA-binding protein with ZF domain. Involved in nucleo-cytoplasmic shuttling of another RNAbinding protein STAU2 in neurons. Related pathways of Diurnally regulated genes. Involved in ER network formation, remodelling and shaping. Involved in mitochondrial heme biosynthesis, as a part of the iron sulfur cluster machinery in mitochondria Infantile onset HSP with 13 marked spasticity and contractures of tendo-achilles, Borderline IQ, rarely associated with nephrotic syndrome. Pure HSP with TCC. 13 Presented as AD or AR pure HSP with urinary sphincter disturbance, pes cavus and rarely mild postural tremors. Early onset slowly progressive HSP associated with axonal PN, pes cavus, visual field defects, optic atrophy. Reduced activity of mitochondrial respiratory chain complexes I and II X Linked SPG1/ Xq28/ L1CAM [L1 cell adhesion molecule] Neural cell adhesion molecule (NCAM) It is an axonal glycoprotein of the immunoglobulin superfamily. Cell adhesion molecule involved in neurite outgrowth, neuron to neuron adhesion, neuronal survival, & development of cerebral cortex. Infantile onset neurodevelopmental HSP. Phenotypic manifestations described under acronyms MASA syndrome [Mental retardation, Adducted thumbs, Shuffling gait, Aphasia] or CRASH syndrome (Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spastic paraplegia, and Hydrocephalus). HSP severe spasticity, dysmorphic features, pes cavus, kypho-scoliosis. NI: TCC, dilated ventricles 3,

16 SPG2/Xq22/ PLP1 SPG22/Xq13/ SLC16A2 (MCT8) SPG16/Xq11/ {locus} SPG34/ Xq24-25/ {locus} Myelin proteolipid protein1 (MPLP1) Monocarboxylase transporter 8 Myelin formation, maintenance of myelin sheath and axonal survival A membrane protein involved in thyroid hormones transport and assumed to have an important role in central nervous system development. HSP with marked progressive spasticity, scissoring resembling CP, MR, cerebellar ataxia signs, nystagmus, optic atrophy. NI: severe lack of myelination. HSP associated with neonatal hypotonia evolved into spasticity, MR, cerebellar ataxia, microcephaly, dysmorphic features including marfanoid habitus & pectus excavatum, multiple joints contractures, dystonia, amyotrophy, thyroid hormones disturbances. NI: mild hypomyelination. - - Very early onset complex HSP with MR, developmental delay, transient nystagmus, short and thick phalanges, maxillary hypoplasia. - - Pure phenotype of HSPs with late childhood onset. 3, , Mitochondrial {MT} MT-TI gene mutation (m.4284g-a) MT-ATP6 gene mutation (m.9176t>c) MT ND4 gene mutation (m.g11778a) MT-CO3 gene mutation (9537insC) mtrna-isolucine transfer [RNAmitochondrial isoleucine] Complex V, ATP synthase subunit NADH ubiquinone oxidoreductase Cytochrome C Oxidase (COX), Subunit III transfer RNA function in mitochondria Synthesis of ATP 6 subunit of MT-complex V Functions in ND4 subunit of MT-complex I Functions in COX complex IV Complex HSP with progressive ophthalmoplegia, cerebellar ataxia, MR, associated cardiomyopathy, hearing loss, diabetes. Dominant HSP with axonal neuropathy, normal lactate, reduced ATP synthesis, cerebellar signs, associated cardiomyopathy Adult onset HSP, painful spasticity, associated with visual loss and sphincter disturbance. Childhood onset HSP with MR, ophthalmoplegia, severe lactic acidosis, enzyme (COX) deficient activity measured in skin or muscle biopsy

17 Unclassified syndromic HSPs AR Cerebral Palsy spastic paraplegia Type 1 GAD1 gene/2q [L glutamate decarboxylase 1], AD Spastic Paraplegia with DNM2 mutation DNM2 gene/19p AR FARS2 gene associated HSP/ FARS2 gene/6p Glutamate decarboxylase 67-KD [GAD67] brain isoform Dynamin 2, large GTPase Mitochomdrial phenylalanyltrna synthetase Conversion of glutamic acid into gamma-aminobutyric acid functions in centrosome, intracellular membrane trafficking, endocytosis, interacts with actin and microtubules, involved in retrograde motor transport Functions in attachment of mt-phenylalanine to its trna Complex HSPS with marked spasticity, MR, microcephaly, scoliosis, multiple contractures, seizures. Variable onset, slowly progressive HSP with pes cavus. Rarely with dysarthria, urinary urgency and mild MR Pure phenotype HSP 137 AD TUBB4A gene associated HSP/TUBB4A gene/19p Beta tubulin Formation of brain specific microtubule. Complex slowly progressive HSPs associated with cerebellar ataxia. NI: hypo-myelination 138 AR AR AR AR MAG gene associated HSP (SPG75)/MAG gene/19q KLC4 gene associated HSPs/KLC4 gene/6p Spastic paraparesis, optic atrophy, polyneuropathy syndrome (SPOAN)/KLC2 gene/11q Infantile Ascending spastic paralysis (IAHSP)/ALS gene/2q Myelin associated glycoprotein Kinesin light chain 4 Kinesin light chain 2 Alsin protein Small GTPase Rab5 Myelin formation Microtubule association as a motor transport molecule Microtubule association as a motor transport molecule Intracellular endoplasmic trafficking. NI: variable cortical atrophy. Complex HSP with cerebellar ataxia, ID, amyotrophy. Complex HSP with joints contractures and remarkable NI abnormalities: TCC, global cortical and cerebellar atrophy, periventricular excessive WM lesions, marked high signal at corticospinal tracts bilaterally. Complex infantile onset progressive HSP with nonprogressive optic atrophy, fixation nystagmus, hyperhidrosis, scoliosis, joint contracures, distal amyotrophy and sensorimotor polyneuropathy [later onset]. Infantile complex HSP progressed into quadriplegia with dysphagia, pes cavus, abnormal eye movements,

18 AR AR AR Spastic paraplegia with EXOSC3 gene mutations/ Exosome gene/9p Spastic paraplegia associated LYST gene mutations/ LYST gene/1q Spatic paraplegia associated with BICD2 gene mutations/ BICD cargo adaptor2 gene/9q Exosome component 3, part of the protein complex RNA exosome Lysosomal transport protein regulator Motor adaptor protein Degradation of un-needed RAN molecules. Important for normal development of the cerebellum and spinal cord motor neurons Lysosomal trafficking and vesicular transport protein regulator Involved in dynein mediated vesicular and mrna transport Complex HSP with mild MR, cerebellar ataxia, strabismus, distal amyotrophy, adducted thumb, atrophy of tongue, short stature. NI: cortical atrophy, vermal cerebellar hypoplasia, enlarged cisterna magna Complex HSP with cerebellar ataxia, demyelinating sensorimotor neuropathy. NI: cerebellar and spinal cord atrophy. Infantile onset complex HSP associated with amyotrophy ER: Endoplasmic reticulum; ERAD: ER associated degradation; NI: Neuroimaging; ID: Intellectual disabilities; MR: Mental retardation; PN: Peripheral neuropathy; CP: Cerebellar palsy; IQ: Intellectual quotient; DD: Developmental delay, TCC: Thin corpus callosum; WM: White matter. Colored circles with number: chromosome number for each gene or locus. Symbol: amyotrophy phenotypic association. Differential Diagnosis of HSPs The particular diseases, acquired or inherited, with a clinical picture that resembles the HSPs of lower limbs spasticity, brisk reflexes, hypertonia, with or without ataxia should be excluded (Table 2). In clinical practice, CNS and spinal cord structural, neurodegenerative, and autoimmune causes (see Table 2) usually are taking the first place of exclusion. The metabolic diseases are usually associated with particular clinical findings that can ease the clinical exclusion. 18

19 Table 2: Differential diagnosis of HSPs. Acquired Myelopathies Neuroinfectious& Structural changes autoimmune Metabolic Disorders - Peroxisomal disorders(adrenoleukodystrophy) Inherited Other neurodegenerative diseases - Cerebral palsy - Cervical spine degeneration - Atlanto-axial subluxation - Chiari malformation - CNS tumor - Vascular causes; spinal cord infarction, duralarteriovenous malformation - Myelitis - HIV - Syphilis - HTLV1 - Multiple sclerosis - Lysosomal diseases(gm1/gm2 gangliosidosis, Krabbe disease, Gaucher s disease, metachromatic leukodystrophy) - Urea cycle disorders(arginase deficiency) - Homocysteineremethylationdefects (Methylene-tetrahydrofolatereductase (MTHFR) deficiency, cobalamin Cdeficiency) - Dobamine synthesis defects - Abetalipoproteinaemia - Biotinidase deficiency - Vitamine B12 (subacute spinal cord degeneration) & E deficiency - Spastic ataxias - Motor neuron disease (Primary lateral sclerosis, familial amyotrophic lateral sclerosis) -Recessive Spinocerebellar ataxias. - Inherited dementias (PSEN1-related disorders). Table references [ ]. Clinical investigations Brain and spinal cord imaging should be done as a routine to exclude neuro-structural causes or specific leukodystrophies. Brain imaging is also very important to reveal HSPs associated CNS anomalies. Plasma amino acids, extended metabolic screening, very long chain fatty acids, lipoprotein profile, serum copper, vitamin E, cobalamin and homocystine are of the clinical investigations recommended in the differential diagnosis, particularly in sporadic cases. Lumber puncture and CSF analysis should be considered (suspect viral infection or to exclude multiple sclerosis). HSPS GENETICS HETEROGENEITY HSPs comprise a markedly genetically heterogeneous group of disorders. This heterogeneity is accounted for in different ways: HSPs inheritance pattern can be Mendelian, sporadic, or rarely of maternal trait. The three modes of Mendelian inheritance were observed among the various SPGs subgroup; for autosomal recessive inheritance 49 forms of AR-SPGs for the dominant, up to date 20 forms of AD-SPGs and HSPs 5 SPGs of the X-linked HSPs, were described in the literatures. Recent reports showed that mutations in same SPGs gene can produce a particular SPG subgroup segregated in different modes of inheritance, examples; SPG3A is known to be of AD inheritance, due to heterozygous mutations in ATL1, was also found to show in a recessive mode with mutations in the same gene [8,50]; SPG30 with KIF1A mutations described both in a recessive pure SPG30 and in a de-novo complex dominant SPG30 [8,102]; SPG72 described with dominant mutations in REEP2 (causing dominant negative effect on the wild encoded protein) as well as with recessive mutations leading to protein loss of functions [8,122]. 19

20 On the other side mutations in the same gene can produce two different phenotypes, even in the same family, suggesting a phenotypic spectrum of the same gene when mutated. Example; KIF5A mutation in a patient clinically presented by axonal CMT peripheral neuropathy was found to be a member of an HSP family (SPG10). This anticipated that SPG10 and CMT may present a phenotypic spectrum resulting from KIF1A mutations [153]. Complexity of HSPs genetics is not only due to different genetic inheritance patterns that are impacting the recurrence rate or the anticipated disease progression, but also it is the HSPs contributing genes, which constitute a long and still growing list. These genes are heterogeneous both in their functions and metabolic impacts, however might interact on a similar or networking, pathways [13]. Sporadic HSP case might presents either a de-novo mutation happens in the germ-line of the affected patient or it is actually a paternal inherited case, however with a negative family history. When the family has only carriers of recessive alleles or harboring dominant alleles of reduced penetrance then inherited Sporadic cases can possibly encountered. The SPGs genes frequently encountered in sporadic HSPs cases, however as well as in familial cases involve SPG 11, KIF5A, SPG7, SPAST, CYP7B1, and to less extent REEP1 and ATL1 [8,2]. Genetic Diagnosis/Genomic Testing Exclusion of HSPs differential diagnosis is the first line in HSPs professional diagnosis. Taking into consideration the extensive clinical and genetic heterogeneity of various HSPs subgroups and the reported association of lower motor neuron signs, cerebellar ataxia, and even head circumference s abnormalities; microcephaly or macrocephaly, which evolved as prominent findings in some SPGs (Table1), consequently the HSPs phenotype delineation is rather difficult. Therefore, the genetic testing tools constitute a mandatory requirement in the diagnosis of HSPs. Genetic testing using HSPs panel for known genes of AD and AR SPGs can be used as a first approach to genetic testing. However, the need of continuous update of this panel with the newly discovered genesas well as the progressive price reduction of the large-scale clinical whole exome sequencing (WES) places WES as the first choice when considering HSPs genetic testing. WES sequencing enables the identification of pathogenic, likely pathogenic variants in coding regions of all the human genes. The proportion of intronic, non-coding genetic mutations contribution to HSPs phenotypes remain to be considered, however it can be worked out only through research studies applying Whole Genomic Sequencing (WGS). HSPs due to mitochondrial genome related mutations; MT-ATP6 or MT-TI could account for some cases, even few, with pure or complex forms of SPGs. These cases can be overlooked, particularly with the notable intra-familial variability in clinical presentations depending on the tissues affected or rate of heteroplasmy. Paying attention to presentations indicating 20

21 mitochondrial involvements is of value in directing the genetic testing to involve mitochondrial genome. MOLECULAR AND POTENTIAL PATHOPHYSIOLOGY MECHANISMS LEADING TO HSP S GROUP OF DISORDERS Degeneration of axons of the long corticospinal tract, the main pathology in HSPs starts at the most distal parts of the axons (distal axonopathy) in a retrograde fashion toward the cell body. HSPs is branded as a clinically and genetically heterogeneous group of diseases for which a large and growing list of genes is expanding. Those genes when mutated, encoding altered proteins that contributing to corticospinal tract degeneration and leading to a wide range of associated abnormal clinical and neuroimaging features. Proteins encoded by such list of genes are found either to interact on different molecular pathways, or act under the same functional category or involved in multiple functions such as the Spastin. Maintenance of corticospinal tract s axonal s integrity requires multiple-functioning and interacting complex machineries to achieve the following: - Maintain adequate levels of mitochondrial ATPase produced energy, - Protect axonal environment against excessive oxidative stress, - Keep normal shaping and morphogenesis of organelles (ER and Golgi) to enable their active functions in clearance of unwanted cargos, - Act in severing/disassembly of microtubules; the axonal cytoskeleton structure, to facilitate its organization and dynamic functions in axonal transport. Microtubule dynamics is coupled to the moving motor molecules; kinesin, dynein, & actin in transporting protein cargos and vesicles along the long corticospinal tract axons in anterograde and retrograde directions, - Maintain proper myelination process with maintenance of myelin sheath, - Promote axonal development, and to - promote autophagy (removing of unwanted cellular components). Based on the above, SPGs proteins were grouped into a number of functioning categories or pathways, which were assumed to interact or networking together. The main functional groups, listed below, form the basis of the updated pathophysiological mechanisms underlying HSPs subgroups: Axonal Development X linked SPG1 and SPG22 are of the known SPGs to play a crucial role in central nervous system and axonal development. NCAM (L1CAM), involved in X-linked SPG1, is a glycol-transmembrane protein that is expressed mainly in neurons and Schwann cells. It plays an important role in the development of 21

22 the nervous system through its interactions with other cell adhesions molecules (CAM) partners, promoting neuron-neuron adhesion and axon outgrowth [154]. SLC16A or MCT8 gene (clinical disease s name Allan-Herndon syndrome or X-linked SPG22), MCT8 encoded protein, monocarboxylase transporter 8 is expressed in cerebral microvesels and neurons. It functions in shuttling thyroid hormones from blood- cerebrospinal fluid (CSF)-blood and in efflux of thyroid hormones inactive metabolites from CSF to blood and is described to lead to axons impairment when mutated [155]. Myelination Impairments PLP1gene (X-linked SPG2) encodes the myelin proteolipid protein (MPLP). This protein comprises the major myelin protein that is responsible for stabilization and maintenance of the myelin sheath [3,125]. FA2Hgene, (SPG35) encodes an NADPH-dependent mono-oxygenase, which is involved in synthesizing 2-hydroxy fatty acids, which is further incorporated in the biosynthesis of sphingolipids and ceramide. Ceramide is of the major consitutents of myelin. FA2H encoded protein is highly expressed in oligodendrocytes [37,156] and have a role in maintaining interactions between sphingolipids and cells membrane to membrane interactions [157]. SPG42/SLC33A1 encodes the acetyl-coa transporter, a multiple trans-membrane protein in the ER. It carries acetyl-coa into the lumen of Golgi apparatus, where it is transferred to the gangliosides and glycoproteins. This gangliosides and glycoprotein modification presumed to play a critical role in motor neurons axons maintenance and outgrowth. Inadequate supply of acetyl- CoA, caused by a reduced flow of acetyl-coa into the Golgi apparatus, can result in misprocessing of gangliosides and glycoproteins [79]. GJC2 (SPG44) encoded protein plays an important role in central and peripheral nervous system myelination. Gene mutations disrupt the junction channels between astrocytes and oligodendrocytes causing cells communication impairment and impaired myelin maintenance [41]. Axonal Transport SPG10 (KIF5A), SPG30 (KIF1A) and SPG58 (KIF1C) is three HSPs associated with kinesin defects. Kinesin are motor molecules involved in anterograde transport of neurofilaments subunits, membrane vesicles, and other anterograde cellular cargos along its interaction with microtubules cytoskeleton [9,158,159]. SPG4 (SPAST) acts to severing (breaking) the microtubules promoting their dynamics in axonal transport. Spastin has multiple isoforms with rather complex subcellular localizations and hence its functions. Spastin localization at the ER, ER-Golgi compartment, cytosolic pool movable to the endosomes, in the nucleus, and to microtubules was described. Spastin interacts with protrudin promoting neurite extenstion [9,160]. 22

23 Membrane and Endosome Trafficking/ Organelle Shaping SPG3 (ATL1), SPG31 (REEP1), SPG12 (RTN2) and the interacting protein Spastin performing in shaping/morphogenesis of ER tubules membrane structures and Golgi apparatus and in formation of ER networks. Atlastin, in particular regulates ER-tubules fusion. In general, ER morphology is determined by microtubules dynamic which is dependent on spastin, interaction between spastin REEP1 and altastin promote ER-networks formation. Vesicles trafficking across ER and Golgi is altered when mutations occur in these genes [9,161,162] SPG11 (Spatacsin) and SPG15 (Spastizin ) interacting together and colocalize with the proteins functioning in trafficking vesicles, ER-shaping and in microtubules dynamics [163]. Adaptor protein (AP) complexes are ubiquitously expressed in tissues. SPG48 (AP5Z1) and AP4 complex subunits; SPG 47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1), SPG52 (AP4S1) mediate secretory vesicles formation and trafficking across Golgi compartment and endosomelysosome system. The AP4-mediated endosomal trafficking presumed to be involved in brain development and functioning [43,164]. Bone Morphologic Protein (BMP) Signaling Atlastin-1 (SPG3), NIPA1 (SPG6), spastin (SPG4) and spartin (SPG20) act to inhibit the bone morphogenetic protein (BMP) signaling [165]. Loss of function of these proteins leads to up-regulation of BMP pathway, which derive the assumption that the regulation of BMP signaling might be a common pathogenic mechanism in these proteins related HSPs. Interestingly, in zebrafish development, it was found that Atalstin1 regulates the axons architecture by downregulating the BMP, loss of function mutation in Atlastin1 leads to severe axonal defects [166]. NIPA1 is expressed in early endosomes and cell surface and found to be a direct binding partner with Atlastin1 [165,167]. Oxidative Stress/Mitochondrial Related Functions Oxidative stress as a contributing mechanism in HSPs pathogenesis was primarily identified in SPG7 mutations. Paraplegin (encoded by SPG7) was found to form a complex with AFG3L2, which is maaa protease important for mitochondrial functions. Paraplegin forms a complex with AFG3L2 at the inner mitochondrial membrane [168]. This complex plays a key role on the oxidative-phosphorylation pathway and forms a kind of quality control over the inner mitochondrial membrane s proteins [169,170]. Increased sensitivity to oxidative stress and impaired function of respiratory chain complex I were reported in SPG7 patients [171]. REEP1 (SPG31), localized to mitochondria and suggested to have a role in protecting the cells against oxidative stress. When mutated, mitochondrial chain complexes I and IV dysfunctions was described in SPG31 patients [172]. 23