Franca Cambi

Hereditary spastic paraplegia: Advances in genetic research

Hereditary spastic paraplegia (HSP) is a diverse group of inherited disorders characterized by progressive lower-extremity spasticity and weakness. Insight into the genetic basis of these disorders is expanding rapidly. Uncomplicated autosomal dominant, autosomal recessive, and X-linked HSP are genetically heterogeneous: different genes cause clinically indistinguishable disorders. A locus for autosomal recessive HSP is on chromosome 8q. Loci for autosomal dominant HSP have been identified on chromosomes 2p, 14q, and 15q. One locus (Xq22) has been identified for X-linked, uncomplicated HSP and shown to be due to a proteolipoprotein gene mutation in one family. The existence of HSP families for whom these loci are excluded indicates the existence of additional, as yet unidentified HSP loci. There is marked clinical similarity among HSP families linked to each of these loci, suggesting that gene products from HSP loci may participate in a common biochemical cascade, which, if disturbed, results in axonal degeneration that is maximal at the ends of the longest CNS axons. Identifying the single gene defects that cause HSPs distal axonopathy may provide insight into factors responsible for development and maintenance of axonal integrity. We review clinical, genetic, and pathologic features of HSP and present differential diagnosis and diagnostic criteria of this important group of disorders. We discuss polymorphic microsatellite markers useful for genetic linkage analysis and genetic counseling in HSP. NEUROLOGY 1996;46: 1507-1514

Proteolipid protein is necessary in peripheral as well as central myelin

Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are major components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.

Novel mutations in spastin gene and absence of correlation with age at onset of symptoms

Article abstract Autosomal dominant hereditary spastic paraplegia is genetically heterogeneous, with at least five loci identified by linkage analysis. Recently, mutations in spastin were identified in SPG4, the most common locus for dominant hereditary spastic paraplegia that was previously mapped to chromosome 2p22. We identified five novel mutations in the spastin gene in five families with SPG4 mutations from North America and Tunisia and showed the absence of correlation between the predicted mutant spastin protein and age at onset of symptoms.

Differentiation of glial cells and motor neurons during the formation of neuromuscular junctions in cocultures of rat spinal cord explant and human muscle

Motor axons extending from embryonic rat spinal cord explants form fully mature neuromuscular junctions with cocultured human muscle. This degree of maturation is not observed in muscle innervated by dissociated motor neurons. Glial cells present in the spinal cord explants seem to be, besides remaining interneurons, the major difference between the two culture systems. In light of this observation and the well documented role of glia in neuronal development, it can be hypothesized that differentiated and long‐lived neuromuscular junctions form in vitro only if their formation is accompanied by codifferentiation of neuronal and glial cells and if this codifferentiation follows the spatial and temporal pattern observed in vivo. Investigation of this hypothesis necessitates the characterization of neuronal and glial cell development in spinal cord explant–muscle cocultures. No such study has been reported, although these cocultures have been used in numerous studies of neuromuscular junction formation. The aim of this work was therefore to investigate the temporal relationship between neuromuscular junction formation and the differentiation of neuronal and glial cells during the first 3 weeks of coculture, when formation and development of the neuromuscular junction occurs in vitro. The expression of stage‐specific markers of neuronal and glial differentiation in these cocultures was characterized by immunocytochemical and biochemical analyses. Differentiation of astrocytes, Schwann cells, and oligodendrocytes proceeded in concert with the differentiation of motor neurons and neuromuscular junction formation. The temporal coincidence between maturation of the neuromuscular junction and lineage progression of neurons and glial cells was similar to that observed in vivo. These findings support the hypothesis that glial cells are a major contributor to maturity of the neuromuscular junction formed in vitro in spinal cord explant–muscle cocultures. J. Comp. Neurol. 438:239–251, 2001.

Changes in the activity of cdk2 and cdk5 accompany differentiation of rat primary oligodendrocytes

Oligodendrocytes, the myelinating cells of the central nervous system, are terminally differentiated cells that originate through asynchronous waves of proliferation and differentiation of precursors present at birth. Withdrawal from cell cycle and onset of differentiation are tightly linked and depend on an intrinsic program modulated by the action of growth factors. p27 plays a central and obligatory role in the initiation of oligodendrocyte differentiation and cessation of proliferation. In this paper, we have characterized the role of modulation of cdk2 and cdk5 kinase activity during the process of oligodendrocyte precursor differentiation. As rat primary oligodendrocytes differentiate in culture there is a fall in cdk2 activity and a rise in cdk5 activity as well as an increase in the cdk inhibitor, p27 protein. The decline in cdk2 activity is not accompanied by a drop in cdk2 protein level, suggesting that it results from inhibition of cdk2 activation rather than decreased protein expression. Taken together, these data suggest that oligodendrocytes may withdraw from the cell cycle at G1‐S transition through inactivation of cdk2 activity, possibly initiated by increasing amount of p27, and that cdk5 may have a role until now unrecognized in the differentiation of oligodendrocytes. J. Cell. Biochem. 68:128–137, 1998.

A PLP splicing abnormality is associated with an unusual presentation of PMD.

We report that a deletion of 19 base pairs (bp) in intron 3 of the proteolipid protein (PLP/DM20) gene causes a neurological disease characterized by mild developmental delay, followed by progressive decline of acquired motor and cognitive milestones. The clinical features are associated with mild delay in myelination demonstrated by magnetic resonance imaging studies and with ongoing demyelination and axonal loss demonstrated by magnetic resonance spectroscopy. We demonstrate that the purine‐rich 19bp element regulates PLP‐specific splice site selection in transient transfections of chimeric constructs into cultured oligodendrocytes. Runs of 4 and 5 Gs centered in the 19bp element are critical for efficient PLP‐specific splicing. The intronic element is sequence specific in oligodendrocytes and is not a repressor of PLP‐specific splicing in nonglial cells. These data support the conclusion that deletion of the 19bp purine‐rich region in PLP intron 3 causes a reduction in PLP message and protein, which affects myelin stability and axonal integrity.

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin‐dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus‐expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur. J. Cell. Biochem. 76:270–279, 1999.

Peripheral Neuropathy Caused by Proteolipid Protein Gene Mutations

ABSTRACT: Pelizaeus‐Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system typically caused by duplications or missense mutations of the proteolipid protein (PLP) gene. Most investigators have found that peripheral nerve function and structure is normal in PMD patients. We have found that null mutations of the PLP gene cause demyelinating peripheral neuropathy, whereas duplications and a proline 14 to leucine mutation do not affect nerve function. A family with a nonsense mutation at position 144, which affects only PLP but not the alternatively spliced gene product DM20, has a very mild syndrome, including normal peripheral nerve function. Our findings suggest that DM20 alone is sufficient to maintain normal nerve function and that there may be domains of PLP/DM20 that have a relatively more active role in the peripheral nervous system compared with that in the central nervous system.

Down-regulation of the retinoblastoma protein (rb) is associated with rat oligodendrocyte differentiation.

Terminal differentiation of oligodendrocytes is associated with permanent withdrawal from the cell cycle. We studied the expression of the retinoblastoma protein, expression and activity of G1 cyclins and kinases in oligodendrocyte progenitor cells cultured in vitro. We found that Rb stopped to be expressed concomitantly with the activation of CNPase in oligodendrocytes differentiated with thyroid hormone. In contrast, Rb continued to be expressed at reduced levels in oligodendrocytes that were arrested in G1 by removal of mitogens. Cyclin D1, cdk2, and cdk4 kinase activities were decreased in G1-arrested and differentiated oligodendrocytes. Cyclin E, however, continued to be expressed in G1-arrested oligodendrocytes. Inhibition of differentiation induced by mitogens in oligodendrocytes arrested in G1 by Ad-p27 was accompanied by continued expression of Rb, D1, and E cyclins. After removal of mitogens and addition of thyroid hormone, Rb stopped being expressed and CNPase expression was activated with a temporal course similar to that of oligodendrocytes infected with a control adenovirus. Our results indicate that Rb may play an important function in differentiation of oligodendrocytes in response to external mitogens and differentiation factors.

GM1 ganglioside in Parkinson's disease: Pilot study of effects on dopamine transporter binding

OBJECTIVE GM1 ganglioside has been suggested as a treatment for Parkinsons disease (PD), potentially having symptomatic and disease modifying effects. The current pilot imaging study was performed to examine effects of GM1 on dopamine transporter binding, as a surrogate measure of disease progression, studied longitudinally. METHODS Positron emission tomography (PET) imaging data were obtained from a subset of subjects enrolled in a delayed start clinical trial of GM1 in PD [1]: 15 Early-start (ES) subjects, 14 Delayed-start (DS) subjects, and 11 Comparison (standard-of-care) subjects. Treatment subjects were studied over a 2.5 year period while Comparison subjects were studied over 2 years. Dynamic PET scans were performed over 90 min following injection of [(11)C]methylphenidate. Regional values of binding potential (BPND) were analyzed for several striatal volumes of interest. RESULTS Clinical results for this subset of subjects were similar to those previously reported for the larger study group. ES subjects showed early symptomatic improvement and slow symptom progression over the study period. DS and Comparison subjects were initially on the same symptom progression trajectory but diverged once DS subjects received GM1 treatment. Imaging results showed significant slowing of BPND loss in several striatal regions in GM1-treated subjects and in some cases, an increased BPND in some striatal regions was detected after GM1 use. INTERPRETATION Results of this pilot imaging study provide additional data to suggest a potential disease modifying effect of GM1 on PD. These results need to be confirmed in a larger number of subjects.